MCB Exam 2

Modification on K9 with methyl

Heterochromatin formation, gene silencing Chromosomes are highly condensed= no gene expression

Chromatin remodeling complex can use energy from ATP hydrolysis to catalyze changes in yhe structure of nucleosome

Figure A Hydrolyze ATP and use that energy to temporarily effect interaction between histone and DNA Catalysis of nucleosome sliding, sliding = change in position of nucleosomes which allows DNA to become more available and allows sequences to become available Nucleosome movement by sliding along a DNA molecule exposes sites for DNA binding proteins

TAQ polymerase

First DNA polymerase used in PCR, found in hot spring Optimal temperature = 75 C

FRET: Fluorescence Resonance Energy Transfer

Fluorescent protein Em: emission wavelengths Exc: excitation wavelengths use to study proteins in the cell, can track protein with for example YFP to track it and you can see where it is located FRET: The ability of a higher energy donor fluorophore to transfer energy directly to a lower energy acceptor molecule

Chromatin packing occurs on several levels

Histone H1 pull the nucleosomes together into a regular repeating array 30 nm fiber is organized into loops emanating from a central axis 300 nm fiber is further condensed to from mitotic chromosome Net result= Each DNA molecule has been packaged into a mitotic chromosome that is 10,000 fold shorter than its extended length

Topological isomers

Molecules differing only in linking numbers

Oxidative damage

Most common oxidative damage occurs on guanine on -CH becomes oxidized and becomes 8-oxoguanine, if use for DNA replication base pairs with A. After replication G:C base pair changes to T:A (transversion)

Fluroescence readout

Next improvement to the Sanger sequencing Instead of having to run 4 reactions, put them together. When labeled the different nucleotides, they would be different colors and you could read out DNA sequence

Illumina Sequencing

No separation based on size, sequencing based on size 1. start with DNA that you want to sequence. At ends put common primers. The primers would be meant to match primers physically associated with the slide allowing the DNA to hybridize on the slide which lead to the amplification, generating a cluster of identical DNA molecules 2. Clusters would then be read out with fluorescences. At one segment at a time, introduce the nucleotide and then use laser to read out color of the spot indicating the nucleotide at that position 3. continue to read at each position to get entire DNA sequence in the cluster On a single slide you could read out the sequence of multiple clusters just by looking at the color of the spot as you go through individual cycles of polymerization Can get a lot of spots on a slide and by following the colors at each spot, you can sequence all of the DNA sequences 1. shear DNA into fragments and adaptors are ligated onto ends. 2. fragmented DNA is spread out over a flow cell using adaptors. 3. PCR is used to amplify colonies of these ligated fragments. 4. SBS- sequencing primers are washed over plate 5. Modified fluorescent nucleotides are incorporated by polymerase one at a time. 6. colored nucleotides are visualized with a camera. Increases speed and efficiency of genome sequencing Human genome sequencing 1. Take DNA from patient 2. Fragment their DNA into small pieces 3. Each fragment would land at a specific spot on the slide 4. Amplification and sequence through fluorescences 5. Can put together the person's genome

Telomere

Protect ends Ensure complete replication of ends Contain repeated nucleotide sequences that enable the ends of chromosomes to be replicated Protect the end of the chromosomes from being mistaken by the cell as a broken DNA molecule in need of repair

PCR steps

Step 1: Denature, 95 C, 30 seconds, denature to make single strand and template strand Step 2: Annealing, 50-60 C, 30 seconds, depends on sequence and length of primer (hybridization of primers) - primers need to be in excess and needs to be 18 nucleotides long to ensure correct sequence - primers form complimentary pairs which is why it is important to know sequence at start and at end because will allow you to design primer which will bind Step 3: Elongation, 72 C, 1 kb/min - DNA polymerase doesn't know when to stop so set length DNA polymerase can go. Process: One DNA molecule becomes two

Three unique sequence elements that are required for the maintenance and replication of chromosomes

Telomere Replication origin Centromere

How are we going to identify the interacting proteins for a protein of interest?

1. Affinity purification followed by mass spectrometry identification 2. yeast two-hybrid screen

FISH Steps

1. Prepare short sequence of single stranded DNA that match a portion of the gene the researcher is looking for= probe 2. Label probes by attaching one of a number of colors of fluorescent dye 3. Let probe bind to complementary strand of DNA= hybridization)

CRISPR

Clusters of Regularly Interspaced Short Palindromic Repeats Bacteria have in their chromosomes have sequence of repeats that are interspaced with sequences from viruses. This is called CRISPR. These CRISPR sequences could be acquired immune system in bacteria which would allow sequences from viruses to be integrated and then used later to protect the cell from an infection from that same virus

Histone code is read by

Code reader complex recognize unique type of modification. Protein complex with catalytic activates and additional binding site. Determine how genes in the region will be expressed ------> biological effects (not only determined by the gene but also the marks around genes on histones)

Hydrolytic attack

Common between bond connecting purine and sugar. Leads to lose of purine (depurination). - occurs in adenine and guanine - the DNA of each human cell loses 5000 purines ( A and G) every day also another example is an attack on C, amino group can be attacked and converted into U - about 100 cytosines are converted to urical via deamination

Comparison of the beads on a string form and the 30 nm chromatin fiber

Compact end= tails are positively charged, high level of histone H1, more methylated, not comparable with gene regulation Less compact end, looks like beads on a string= highly acylated core histones, reduced level of histone H1, more comparable with gene expression, less methylation

Higher order structure of chromatin

A model for the structure of a eukaryotic chromosome shows that the majority of the DNA is packed into large loops of 30nm fiber that are tethered to the nuclear scaffold at their base Sites of active DNA manipulation (sites of transcription or DNA replication) are in the form of 10n fiber or even naked DNA

Epigenetic Regulation/inheritance

A process that affects the expression of specific genes and is inherited by daughter cells, but is NOT the result of a change in DNA sequence Gene Y on--- chromatin change---> Gene Y off (highly condensed so cannot be expressed, silenced) ---- multiplication of somatic cells---> 2 gene Y off ( stay inactive not expressed) Chromatin based epigenetic information in genes of eukaryotes serve as a memory

Rare tautomers of DNA bases can cause mispairing

A*: C mispair T*:G mispair G*:T mispair C*:A mispair

Position effects on gene expression---> ADE2

ADE2 gene at normal location on chromosome, gene expressed because far from heterochromatin region ADE2 gene moved near telomere, not effectively expressed

Depurination

AP site= no base attached to sugar AP can be recognized by AP endonuclease to be fixed hydrolytic attack single nucleotide deletion can occur due to depurination

PCR

Amplification of a specific DNA fragment by polymerase chain reaction If you want to amplify that region of DNA need to know sequence at start and at end of DNA of target DNA. Then can use PCR, don't need to know sequence of whole thing

Cockayne Syndrome

Can't prioritize the fixation of the errors in DNA poor growth premature aging sensitivity to sunlight development and neurological delays shortened lifespan

Heterochromatin

Chromatin in the interphase nuclei that exists in a HIGHLY CONDENSED form In mammalian cells, about 10% of the genome is packaged into heterochromatin Usually does NOT contain actively expressed genes Telomere and centromere are examples of heterochromatin region

Euchromatin

Chromatin in the interphase nuclei that is LESS condensed Contain actively expressed genes

cDNA

complimentary to mRNA

Mismatch repair

detecting and binding a mismatched base pair removing the lesion from the newly synthesized strand but NOT from the parental strand - must be able to distinguish two strands repairing DNA synthesis Special proteins required for E.coli - MutS - MutL MutH

MutS

detecting and binding a mismatched base pair (scanning)

Screening natural products or libraries of compounds can yield drugs or drug leads

ex. aspirin, extract from bark which could cure pain and isolated the one component added acetyl group to salicylic acid to make aspirin you convert arachidonate to prostaglandin H2 which triggers the headaches or inflammation = pain Reaction synthesized by prostaglandin H2 synthase Active site has Ser residue on opening of active site once aspirin binds here it can transfer its acetyl group to the side chain of Ser (so is know covalently modified, irreversible inhibitor) so no longer can produce prostaglandin so no headaches

Spontaneous alternations likely to require DNA repair

hydrolytic attack oxidative damage uncontrolled methylation

Deamination

hydrolytic attack sometimes find U in DNA due to damage to C Uracil can be corrected

Methylation allows

identification of templated versus new DNA strands in E.coli replication for a short period following replication, the template strand is methylated and the new strand is not after a few minutes the new strand is methylated (Dam methylase) and the two strands can no longer be distinguished At this point even if detect mismatch, can not change error

TAD

important because relevant genes in similar domain, so easily access to same protein that regulates expression so more efficient

H2A.X

important for DNA repair, replaces H2A in nucleosomes. H2A.X becomes phosphorylated upon DNA break, which helps to recruit DNA repair enzymes to fix DNA double helix

CENP-A (a H3 variant)

important for centromere function Allows the attachment of the mitotic spindle to the kinetochore Long N' terminal end which can interact with kinetochore

Plasmids

in prokaryotes small independent circular DNA, which carries genes that confer desirable traits to the bacteria

Modification K27 with methylate

silencing of Hox genes, X chromosome inactivation found in female

Histone acetyl transferase

Transfers acetyl group from acetyl CoA to side chain of Lys

what do DNA replication, repair and recombination have in common

Utilize similar enzymes All involve the making or breaking of phosphodiester bonds/links

Nonhomologous end joining

Utilizes KU protein which joins ends together have some nucleotides removed before you can join because nuclease works faster than KU protein

Drugs can be

designed on the basis of three-dimensional structural information about their targets poppy flower--> morphine--> upon binding of morphine two responses either pain relief (Gi/o) or unwanted side effects (B-arrestin) with testing and optimization= PZM21 binds and unwanted side effects (beta-arrestin= Shut off) turned off and only pain relief. no side effects

DNA helicases, clamp loader and DNA ligase have what in common

all require energy from ATP input, from ATP hydrolysis

Processive enzyme

an enzyme that catalyzes multiple rounds of the elongation or digestion of a polymer while the polymer stays bound

Nucleosomes prefer to bind bend DNA

5 A:T, 5 G:C, 5 A:T, 5 G:C rich will very easily wrap around histone, preferred sequence because A:T rich region tends to bend toward minor groove and G:C rich region tend to bend toward major groove site so if alternating, DNA will get perfect bend Sequences that alternate between A:T and G:C rich sequences with a periodicity of approximately 5 base pairs will act as preferred nucleosome binding sites

Requires energy from ATP hydrolysis

DNA helicases DNA topoisomerase II Clamp loader DNA ligase He likes the clover

How will hexameter know when you have sufficient nutrients?

DNA hexameter are ATP binding proteins Only when DnaA can bind to ATP can it form hexameter and bind to origin

Why is it important to package DNA into chromosomes

DNA is compacted in the chromosome, so it readily fits inside the cell Packaging the DNA into chromosomes serves to protect the DNA from damage: - completely naked DNA molecules are relatively unstable in cells; in contrast, chromosomal DNA is extremely stable Only DNA packaged into a chromosome can be transmitted efficiently to both daughter cells when a cell divides The chromosome confers an overall organization to each molecule of DNA. This organization regulates gene expression as well as the recombination between parental chromosomes that generates the diversity observed among different individuals of any organism

Direct repair

DNA photolyase - direct cleavage of pyrimidine dimer into its component bases - correct pyrimidine dimer Demethylase - direct removal of methyl groups from methylated bases - corrects wrong methylation

Two ways pyrimidine dimer can be fixed

DNA photolyase (direct repair) Nucleotide excision repair

Prokaryotic polymerases

DNA polymerase I= Erases primer and fills in gaps on lagging strand DNA polymerase II= error prone polymerase, DNA repair, deal with emergency with DNA repair DNA polymerase III= primary enzyme of DNA synthesis

Eukaryotic polymerase

DNA polymerase alpha= initiator polymerase, first 20 nucleotides produced - primase subunit= synthesizes the RNA primer - DNA polymerase unit= adds stretch of about 20 nucleotides to the primer DNA polymerase beta= error prone polymerase, DNA repair, deals with emergency with DNA repair DNA polymerase S= primary enzyme of DNA synthesis DNA polymerase E= primary enzyme of DNA synthesis in leading strand DNA polymerase Y= mitochondrial polymerase

Recombination

Two daughter DNA molecules are formed by the exchange of segment between two parent DNA molecules Generate two new DNA molecules

radiation

UV radiation can lead to formation pyrimidine dimer Ionizing radiation (gamma, x-ray) leads to double strand break

Defects in DNA repair lead to diseases (two examples)

Xeroderma pigmentosum (XP) ----> nucleotide excision repair (use pathway to fix pyrimidine dimer, will always occur because always exposed to UV light but typically repaired right away but if enzyme is defective, it can't be repaired becoming very sensitive to the sun) Werner syndrome-----> accessory 3'-exonuclease and DNA helicase

Benefit of cDNA

You know every gene is intact, you can screen and identify gene of interest in tissue

Genome targeting technology

ZFN TALEN - Both programmable ways to generate double strand breaks but rely on protein based recognition of DNA sequencing. Requires a lot of protein engineering CRISPR/Cas9 - RNA programmed protein, single protein, to be used for any site on DNA that we want to generate break by changing the sequence of the guide RNA associated with Cas9 - Dont rely on protein based recognition. Rely on RNA based recognition

ZFN and TALEN

ZFN: composed of a DNA binding zinc-finger protein domain and the nuclease domain from the Fokl restriction enzyme TALEN: composed of DNA-binding transcription activator-like effectors (TALEs) and the nuclease domain from Fokl BOTH= Use programmable DNA-binding domains (a protein) to direct a nuclease (protein can recognize specific region of DNA)

Purine

1. Adenine 2. Guanine

Mechanisms that ensure the dynamic nature of chromatin structure

1. Chromatin remodeling complex: protein machines that use the energy of ATP hydrolysis to change the structure of nucleosomes - Protein complex hydrolyzes ATP and uses the energy to change interaction between DNA and histone 2. Reversible medication of histones, change charge through post transcriptional modifications of histones

Pyrimidine

1. Cytosine - if hydrolyzed can become uracil which can sometimes spontaneously occur in our bodies, damaging our DNA 2. Uracil 3. Thymine - methylated uracil

Major challenges faced by drug developers

1. Drug candidates must be potent modulators of their targets - if not potent, need high concentration of drug which isn't good or realistic - examine how tightly can bind 2. Drugs must have suitable properties to reach their targets in the body 3. Drugs must not be so toxic that it seriously harms the person who takes it

Finding binding partners: Steps

1. Express affinity tagged protein of interest 2. Form protein complex with its partner 3. Capture the complex using affinity column 4. Separate the complex by SDS-page (dissociates aggregations so we can tell individuals) 5. Reveal the identity of each band on SDS-PAGE by mass spectrometry

Process of homologous recombination overview

1. Introduce a double strand break in one of the DNA molecules by specialized nuclease (produced in meiosis, produced to make double stranded DNA break) 2. Nuclease will degrade it from 5' end, generating single stranded region (3' end stays intact) 3. Single strand region can invade adjacent DNA molecule and start homologous recombination process 4. If found similar base sequence, can base pair with adjacent molecules 5. 3' end acts as a primer and DNA synthesis occurs. Helicase can open up template and polymerase will extend 3' end 6. Eventually form a joined molecule. Use nuclease to cut to make 2 different molecules, cross over occurred

steps for recombinant DNA formation from Sapling

1. Isolation of desired gene and bacterial plasmid 2. both the plasmid and the desired gene are cut using the same restriction enzyme 3. DNA ligase is used to splice together the plasmid and the desired gene at their complementary sticky ends 4. Bacterial cells take up the recombinant DNA plasmid 5. Rapid reproduction of the bacterial cells produce many bacteria that contain the recombinant DNA 6. Mass production of recombinant DNA

Basic steps in the generation of a genomic library

1. Select vector. Isolate genomic DNA and cleave it with restriction enzymes to generate fragments. 2. Cleave vector with restriction enzyme 3. Ligate fragments into vectors= recombinant DNA molecule (each contain unique fragment from the genome) 4. Introduce recombinants into host cells 5. Generate library of hosts, propagate individual cells to produce clones. Library used to locate gene of interest

Three ways in which two proteins can bind to each other

1. Surface string, common for protein kinase and substrate 2. Helix-Helix, coiled coil, common for gene regulatory protein 3. Surface-Surface, most common and specific

PCR Steps Sapling

1. The reaction mixture contains four copies of a particular DNA sequence 2. The reaction mixture is heated to 94 C 3. The double stranded DNA is denatured to form single stranded DNA 4. Temperature of the reaction mixture is lowered to allow the primers to anneal to the DNA template 5. The reaction mixture is heated to 72 C 6. Taq polymerase synthesizes a new DNA strand 7. The reaction mixture contains 8 copies of the DNA sequence

Proteins are the most versatile macromolecule in living systems and serve crucial functions in essentially all biological processes

1. catalyze chemical reactions: enzymes 2. transport: hemoglobin and myoglobin 3. participate in contractionL myosin and actin 4. provide protection: antibodies, interferon and fibrin 5. hormones: insulin, luteinizing hormone, FSH 6. detect and transmit signals: receptors and G proteins 7. control growth and differentiation: transcription factors The biological properties of a protein molecule depend on its physical interaction with other molecules

Two approaches to drug discovery

1. compound ---> physiological effect (ex. reduce blood flow) ---(additional step)-> molecular target Discovery by 1. serendipity/chance observation 2. the fractionation of materials known to have medicinal properties 3. screening of natural products or other libraries of compounds 2. Molecular target--> compound --> physiological effect (test if compound produces effect you predicted and readjust it by giving it to patient or animal) Discovery by: screening or designing molecules with desired properties, that bind to the target molecules and modulate its properties

Tm is affected by what factors

1. concentration of ions in the solution: if high concentration of cations, Tm increases because phosphates tend to repel each other so neutralize their phosphates using cation 2. DNA sequence: if more G-C Tm increases 3. Length of DNA: longer DNA, more H bonds= increase Tm

The structure of nucleosome core particles... extensive interactions

1. hydrogen bonds between backbone of DNA and backbone of histone molecule 2. Charge (- DNA and + histone) 3. Hydrophobic interactions between part of DNA and histone

Function of DnaC

1. load helicase to correct spot. DnaC can interact with initiator protein to make sure correct spot 2. keep helicase in the inactive state

Model that explains how the packaging of DNA in chromatin can be inherited during chromosome replication

1. parental nucleosomes with modified histones 2. only half of the daughter nucleosomes have modified histones 3. parental pattern of histone modification re-established by reader-writer complexes that recognize the same modifications they catalyze (exact same modification=exact same chromosome as parents)

Phase 1, phase 2, phase 3

10-100 subjects safety 100-1000 subjects safety, efficacy, and dosage >1000 safety, efficacy and side effects

Milestones in the Study of DNA

1944 Avery and colleagues show that DNA is responsible for the transmission of heritable characteristics: DNA is the "blueprint" of life 1953 Watson and Crick double-helical structure of DNA - provided mechanism by which DNA blueprint could be replicated every generation 1959-1965 Nirenberg, Khorana and colleagues solve the genetic code - allowed us to take simple sequence of polymer (AGCT) and read out protein it encoded

Distortion of DNA's base paired structure by pyrimidine dimers

2 pyrimidines adjacent to another Exposure of DNA to UV light (sunlight) Double bonds absorb UV light/energy Use that energy to form covalent bond between pyrimidines forming cyclobutane ring length goes from 0.34 nm to 0.16 can lead to kink which can be recognized and repaired if not repaired can impair transcription and replication OVERALL: double bonds in the pyrimidine ring absorb UV light. Adjacent pyrimidine rings form covalent bonds with each other

CRIPSR-Cas9 technology

2-component system for genome engineering cleaving enzyme Relies on RNA-DNA base pairing to determine sites of editing Cas9-mediated editing is efficient, site-specific, can be "multiplexed" - programed Cas9 with multiple different guide RNAs in same cell that can lead to multiple breaks

Solenoid

6 nucleosomes create 1 turn linker DNA so doesn't have to be very long because doesn't have to cross axis

Length of DNA

6.4x10^9 base pairs Each base pair is 0.34 nm (0.34x10^-9 m) so about 2.2 meters long

Imino tautomer C pairs with

A rare Cytosine* H donor

Chromosome

A single large macromolecule of DNA plus the DNA-bound proteins which serve to package and manage the DNA

Cas9 is a

Ability to interact with DNA and generate double stranded break at sequences that match in guide RNA Guide RNA base pairs with one strand of the double helix. RNA interacts with tracrRNA that recruits the Cas9 TracrRNA and Guide RNA are required for this protein to recognize viral DNA/ what they will cut up Link together Guide RNA and tracrRNA to create single guide RNA, making a programmable cleaving enzyme

Cytosines methylated

About 3% of cytosines are methylated in vertebrate DNA and their deamination can NOT be easily repaired C:G becomes T:G (which is hard to identify because natural base) C:G becomes U:G (easier to identify because U is unnatural base) Repairing via a special DNA glycosylase that recognizes a mismatched T-G is relatively inefficient in vertebrates Methylated C nucleotides are common sites for mutations

ADME properties of drug candidates

Absorption (into bloodstream), Distribution (to all places), Metabolism (transformation into metabolites to be excreted) and Excretion The concentration of a compound at its target site is affected by the extents and rates of absorption, distribution, metabolism and excretion If bad ADME properties than you would need a very high dosage of the drug

Capillary electrophoresis

Advancement on Sanger sequencing Separation of DNA by size No need to pour gels and capillaries are much smaller than gel Mixture of DNA labeled with fluorescence, separated by capillary based on size, laser than excited DNA giving you nucleotide sequence based on color and size Able to do this test on human genome by scaling up in a factory but takes a lot of time (bc so huge genome sequence) and money so wanted to find a way to do cheaper

Genomic library

All fragments of genomes useful for scientists to isolate gene of interest can use Recombinate DNA strategies to generate genomic library

Fingers of DNA polymerase

Associates with the template region, leading to a nearly 90 degree turn of the phosphodiester backbone between the first and the second bases of the template This bend serves to expose only the first template base and avoids any confusion concerning which template base should pair with the next nucleotide to be added Can cause conformational change 90 degree turn helps to expose next base pair to make sure no mistake

Different forms of DNA

B form A form Z form

Types of DNA repair systems

Base excision repair - abnormal bases (ex. uracil, removal damaged base) Nucleotide-excision repair - DNA lesions that cause large structural changes (ex. pyrimidine dimers, remove chunk of nucleotides) Mismatch repair (mismatches, fixes mistakes that occur during replication) Homologous recombination of NHEJ (double strand breaks) Direct repair

Summary of DNA replication

Bidirectional and extremely processive Beings at replication origins; each origin generations two replication forks moving in opposite direction One strand (leading strand) is synthesized continuously; another strand (lagging strand) is synthesized discontinuously, in short pieces (Okazaki fragments) Proofreading activity of DNA polymerase corrects polymerization errors Replication machine: cooperative assumably of many proteins in replication fork

Barrier DNA sequences

Block the spread of reader-writer and thereby separating neighboring chromatin domains Mechanism 1. Barrier protein that binds to sequence and stops spread Mechanism 2. Barrier protein enzyme binds to region and erases whatever code is written on the barrier sequence so no spread. Ex. If code is methylation, barrier protein could be de-methylate = stop spread.

Other chromatin binding domains

Bromodomain---> recognizes acetyl-Lys Chromodomain---> recognize methyl-Lys Tudor domain---> recognize methyl-Lys, contact different from PHD example but recognizes trimethylated SANT domain ----> recognize unmodified histone tails

Three steps to acquire immunity in bacteria

CRISPER = acquired immune systems in bacteria 1. Detect foreign DNA that gets injected that gets into the cell 2. CRISPR allows for integration of small DNA molecules from the virus/foreign substance into CRISPR locus 3. CRISPR sequences are transcribed in the cell into RNA. 4. Used to form interference to base pair with matching sequences in viral DNA Take invaders and turn sequence against the invaders

RNA guided nuclease

Cas9, easier because only need RNA Generate guide RNA sequence which will be complimentary of target sequence in genome Guide RNA can bind to complex with nuclease (Cas9) and can target locus depending on base pairing between target sequence and guide RNA Create specific double strand break Cas9= nuclease OVERALL: Recognition of its target DNA sequence relates on a short stretch of RNA (guide RNA) with sequence that is complementary to the target sequence

DNA polymerase definition

Catalyze the elongation of the DNA strand (gets primer)

Responses to DNA damage

Cell cycle checkpoint activation (cells stop cell cycle to repair DNA and then go to next phase of replication) Transcriptional program activation DNA repair Apoptosis (remove bad cells)

Base excision repair

Cell notices error, ex. shouldn't have U in DNA Uracil DNA glycosylase (finds U and cleanse bond) AP site (only sugar and phosphate) AP endonuclease recognizes AP site and phosphodiesterase removes sugar phosphate DNA polymerase adds new nucleotides, DNA ligase seals nick NO HELICASE NEEDED

DNA polymerase continued

Correct base pairing between incoming nucleotide and the template DNA triggers a conformational change in DNA polymerase: a 40 degree rotation of one of the helices in the finger domain = O-helix In the open conformation, the O-helix is distant from the incoming nucleotide; when the polymerase is in the closed conformation, this helix moves and makes several important interactions with the incoming dNTP - A tyrosine makes stacking interactions with the base of dNTP - The two charged residues (Lys and Arg) associate with the triphosphate - The combination of these interactions positions the dNTP for catalysis mediated by the two metal ions bound to the DNA polymerase

Programmed Cas9 cleaves

DNA at specified sites

Sites of contact between the histones and the DNA

DNA bent around histone between minor grove of DNA and histone. Need 10 base pairs to make curve/turn from 1 minor grove to the next The majority of H-bonds are between the histone proteins and the oxygen atoms in the phosphodiester backbone near the minor groove of the DNA

Steric constraints prevent

DNA polymerase form using rNTP precursors DNA polymerase can distinguish between dNTP and rNTP (which is much more abundant in our cells) In second illustration, can not form favorable interaction shifting and misalignment of 2 atoms so reaction will not occur

Basics of DNA synthesis reaction catalyzed by

DNA polymerase which requires 1. template 2. free -OH at the 3' end of a polynucleotide chain (primer), which base pairs with template 3. Uses deoxyribonucleoside triphosphate (dNTP) to extend DNA strand exclusively in the 5'-->3' direction Nucleotide base pairs with template, if base pair is correct it can trigger reaction, which 3' -OH group acts as nucleophile and attacks phosphate atom

Era of personal genomics

Determining who will benefit (or have unacceptable side effects) from a given drug - lower costs of developing drugs and make them more effective - give drugs to those would benefit most Identifying disease genes - whats different from one to a different genome that makes them have a certain disease

Structure of DNA helicase

Diameter of helices large enough for ONE single stranded DNA Can bind to DNA and grab a hold 6 identical subunits, each with ATP binding site. 2 subunits are always empty and 4 are always full with ATP DNA helicase= enzymes that move along and pry apart DNA helix using energy from hydrolysis of ATP

DNA photolyase repair

Direct repair DNA photorylase comes in and fixes damage in adjacent pyrimidines which are covalently linked when exposed to UV light DNA photorylase can recognize this mistake give it light which provides energy, cofactor=FADH which can absorb energy from light and use it to break covalent bond between the two pyrimidines (resolved) Also called photoreactivation repair

DnaA hexamer

DnaA can form hexameter which binds to 5 binding sites in origin Binding of hexameter to origin is highly regulated. Only step in DNA replication in e.coli that is regulated Nutrient status limits it, when sufficient nutrients, hexameter is formed and will bind to origin and start replication of DNA Initiation occurs only when sufficient nutrients are available

Drug metabolism

Drug= foreign compound so our body can try to remove it/metabolize it Body's two main pathways for depending against foreign compounds Phase 1: Oxidation by cytochrome P450 enzymes in the liver Ex. ibuprofen is mainly hydrophobic --oxidation--> enzyme adds hydroxyl group to ibuprofen so increase in hydrophilicity so easy to excrete out. This addition of the functional group allows drug to go to phase 2 of metabolism Phase 2: Conjugation of drug to glutathione, glucuronic acid, and sulfate in the liver (increases hydrophilicity so easier to excrete out. in excretion system specific receptor so conjugation acts as a label making it easier to get it out)

Genome editing begins with

DsDNA cleavage Technology to introduce double stranded breaks at targeted places in the cell. With all of the genome sequencing data, could use this technology to introduce DNA that could fix a mutation or generate a mutation that you want to study so your DNA can repair itself by NHEJ or HR Power: generate double stranded breaks at sites we choose by programming Cas9 and then allow cell to make repairs that introduce genetic changes at sites of these breaks

Nucleosomes are highly dynamic

Dynamic is important because allows access of specific underlying DNA by proteins involved in gene expression, DNA replication and DNA repair

Why can E.coli grows so fast

E.coli can have multiple rounds of DNA replication at similar times. Can't do DNA replication when in hemimethylated. After 15 minutes can start another round of DNA replication

Territory of chromosomes

Each of the 46 interphase chromosomes in a human cell tends to occupy its own discrete, unique territory within the nucleus Well organized FISH

Scientists that made significant contributions to development of CRISPR/Cas9

Emmanuelle Charpentier Jennifer Doudna Martin Jinek Krzysztof Chylinksi Ines Fonfara

Type I DNA topoisomerase

Enzyme utilizes Tyr (catalytic residue) can attack phosphodiester linkage. Leads to formation of new dieter linkage between Tyr and DNA which leads to break in DNA single strand 1. One end of the DNA double helix cannot rotate relative to the other end. 2. DNA topoisomerase covalently attaches to a DNA phosphate, thereby breaking a phosphodiester linkage in one DNA strand 3. The two ends of the DNA double helix can now rotate relative to each other, relieving accumulated strain

exposure to substances in the environment

Ethyl-methane sulfonate (EMS), ethidium bromide change base pairing properties

Limitation to genomic library

Even when you know the gene is present for sure but cannot identify by screening library because when digest gene with restriction enzyme could split gene into fragments so genes would be in different vectors

Proteins and enzymes that are required for DNA replication

Every time we synthesize Okazaki fragment we need to reload sliding clamp so we utilize clamp loader on lagging strand DNA helicase and DNA primate make up primosome

Position effects on gene expression--> white gene

Example with flies White gene at normal location is far away from heterochromatin and barrier. Fly has red eyes White gene close to heterochromatin with rare chromosome inversion where barrier is in place were gene should be leads to red and white eyes of flies

Ejecting nucleosomes and exchanging H2A-H2B dimers by certain chromatin remodeling complex

Exchange of H2A-H2B dimers with either unmodified or variant H2A-H2B dimers, which changes how DNA is excessed Histone chaperone protects histone, typically negative charge DNA lacking nucleosome is very hard to access

Kinetics plays an important role in proofreading SUMMARy

For most DNA polymerases that have exonuclease activity, the exonuclease site is far rom the polymerase site. Thus, several base pairs must be unwound to move the 3'-OH terminal nucleotide in the daughter strand from the polymerase site to the exonuclease site Misalignment makes a mismatched 3'-terminal nucleotide a poor substrate, thus extension from a mismatch is very slow. The delay in extension gives time for partial unwinding of the template-primer, a process that places the primer in the exonuclease site Thus, the 3' exonuclease activity has no specificity fo a mismatched nucleotide; rather, a mismatched nucleotide has a far higher probability of reaching that site than does a matched nucleotide

Holliday junction

Formed during homologous recombination Cross strand exchange: a special DNA intermediate that contains four DNA strands and two of those strands are crossed over When resolved can lead to - no crossover by resolving via crossed strands - crossover by resolving via non crossed strands Depends on which strand resolved, then the product of homologous recombination will be different

Reagents need for PCR

Four nucleotides Taq polymerase/thermal stable polymerase template DNA strand two primers

Amino tautomer C pairs with

G Normal Cytosine

DNA double helix

G and C, and A and T base pairing has exact same shape so way for cell to detect any damage G and C always 3 H bonds A and T always 2 H bonds 10 base pairs for each complete turn of helix (3.4 nm) and bases are about 0.34 nm away from each other. Leadings to base stacking forces which stabilizes the DNA strands (noncovalent interactions)

Major Groove Site

G:C---- A A D H C:G---- H D A A minor grooves for both = ADA (very hard to distinguish if only look at minor groove) A:T---- A D A M T:A---- M A D A minor grooves for both = AHA There are characteristic patterns of H-bonding and of overall shape that are exposed in the major groove that distinguish each base pair These patterns are important because they allow proteins to unambiguously recognize DNA sequences without having to open and thereby disrupt the double helix Thus, proteins that recognize specific DNA sequences (such as transcription factors) tend to bind DNA at its Major Groove

G1

Gap the longest part of the cell cycle main period of cell growth

Modification on S10 with phosphate or K14 with acetylate

Gene expression Phosphorylation adds negative, so less interaction with DNA so open up Acetylate removes positive so more negative

Genetic inheritance

Gene x on---DNA sequence change/mutation---> gene X off----multiplication of somatic cells----> 2 gene X off expressed

Protein protein network in cell

Guilt by association: 1/3 proteins we have no idea what they do/their function we can see what proteins it interacts with and that could help determine the function of the unknown protein

What are the forces that drive the formation of 30 nm chromatin fiber

H1= linker histones which 2 binding sites. H1 can bind to DNA or linker region high concentration of H1 can promote compact of chromatin region

Histones: proteins that pack DNA

H2B, H2B, H3, H4 are responsible for folding DNA into nucleosomes (core histones) - Lys or Arg rich (very basic, +) H1 is bound to the DNA where the double helix enters and leaves the nucleosome core (linker histone) binds to linker region - Lys rich Histones are highly conserved among species. Through evolution, sequences rarely changes. Serve such a fundamental basic function you need every residue to be perfect

Assembly of a histone octamer

H3, H4 dimers interact to form H3, H4 tetramer which is very stable and won't dissociate H2A, H2B form dimer All come together to form histone octamer, DNA wrapped around and tails of the dimers are sticking out

Covalent modification of core histone tails

HAT (Histone acetyl-transferase): opens chromosome structure, activator. Makes more negative so doesn't attach to negative DNA as much Histone Methyl Transferase: will transfer activating methyl from methyl donor on histone, tighter packing= no gene access Histone de-acetylase Histone de-methylase

Ubiquitin

Has many lysine and free N' terminal NH2 group so ubiquitin in itself can become ubiquitinated so possible to build ubiquitinated chain on substrate

Covalent modifications produce

Histone code that helps to determine biological function 1. Combination of various modifications that occur on histones can generate thousands of modification patterns on nucleosomes 2. Many modification patterns on nucleosomes appear to have a specific meaning for the cell (modification determine how gene is expressed in that region) 3. Some of the modification patterns on nucleosomes can be inherited after cell division Covalent modification on histone tail (Mark)

Double helix can be reversibly melted down

Hypochromism: stacked bases in nucleic acids absorb LESS ultraviolet light than do unstacked bases. Melting temperature (Tm): the temperature at which half helical structure is lost. - High Tm= stronger double helix= more stable

Administration and Absorption of drug

Ideal: A small tablet taken orally (absorbed from gut into bloodstream) Lipinski's rules: poor absorption is likely when: 1. the molecular weight is greater than 500 2. the number of H-bond donors is greater than 5 (polar so hard to go through membrane to absorb) 3. the number of H-bond acceptors is greater than 10 (polar so hard to go through membrane to absorb) 4. the partition coefficient (i.e., log(P)) is greater than 5 (very very non polar and would tend to aggregate so hard to get out of membrane after it has crossed) Partition coefficient: measure the tendency of a molecule to dissolve in membrane, how easy molecule can cross membrane log(P): logbase10 of the ratio of the concentration of a compound in an organic phase, n-octanol, to the concentration of the compound in water

chromosome conformation capture

Identify neighborhood of chromosomes 1. Crosslink 2. Restriction nuclease (digests) 3. DNA ligation of cross linked fragments (use ligase to join creating hybrid) 4. Reverse crosslink 5. Sequence DNA (which region of chromosomes are close together. Allows scientists to figure out precisely how each topologically domain contains what type of fragment)

DNA polymerase grips the template and the incoming nucleotide when a correct base pair is made

If correct base pairing, can trigger conformational change alpha helix will close down Tyr will interact with base Lys and Arg (positive charge) interactive with phosphate Interaction helps to position incoming nucleotide close to metal ions and align P- near OH group so reaction can occur

Fully methylated origins

If in GATC sequence, A can be methylated Dam methylase is responsible for methylating all E.coli GATC sequences, allows you to recognize which sequence is parent (methylated) vs newly synthesized (non methylated) Initiation occurs if sufficient resources are available to complete a round of DNA replication. Fully methylated origin---> hemimethylated origins are resistant to initiation (newly replicated DNA unmethylated) for 10-15 minutes --->Origins become fully methylated making them again competent for initiation

How can homologous recombination fix a double strand break

Intact sister chromatids that can guide reaper of double strand break recBCD can recognize double strand break, leading to strand invasion forming of heteroduplex DNA synthesis and migration of branch point (DNA synthesis point is to copy information from sister chromatids and use that copied information to fix damaged occurred) Once enough synthesis, the newly formed elongation strand can go back and base pair with own complimentary strand. Can use copied information to fix damages Use ligase to zip everything back up Allows for very accurate repair

Chromosomes exist in different states throughout the cell cycle

Interphase - begins with extended chromosomes and then ends replicated M phase - condensed chromosome Interphase - segregated and extended

The t-loop

It is a protective structure that prevents the ends of chromosomes from exonuclease activity formed by complementary base pairing of telomeres

Studying protein protein interaction in live cells

Jellyfish fluorescent because has green fluorescent protein so can spontaneously fold into very compact structure and residues can fold at chromophore

DNA synthesis in eukaryotes is more challenging

Large size: 6 billion base pairs in human vs 4.6 million base pairs in E.coli Many origins of replication on each chromosome More chromosomes Chromosomes are linear rather than circular DNA is wrapped around nucleosomes

DNA supercoiling

Lk (linking number)= number of times that a strand of DNA winds in the right-handed direction around the helix axis when the axis lies in a plane Tw (twisting number)= number of times one strand completely wraps around the other strand (not on same plane) Wr (writhing number)= number of times the long axis of the double helix crosses over itself or is wound in a cylindrical manner (how many times supercoil?) Lk=Tw+Wr

Prominent covalent modifications found on histones

Lysine can be modified by adding acetyl group to not have charge, removes positive charge which neutralizes lysine= weaken DNA and histone interaction Serine on histones can be phosphorylated which adds negative charge so histone is more negative= weaken DNA and histone interaction Lysine can be methylated which can preserve the positive charge on Lys= tend to enhance DNA and histone interaction

Zinc finger nucleases

Make fusion protein, nuclease fused with select Zn-finger regions which will each recognize 3-nucleotide sequence. If you know sequence adjacent to target sequence to cut, can identify combination of Zn-finger motifs which will recognize. Done on both sides Dimer than can direct nuclease and generate double strand break Overall: the sequence specificity of ZFNs is determined by ZFPs, each can be designed to recognize a 3-bp DNA sequence

A drug may be toxic

May modulate the target molecule itself too effectively May modulate the properties of proteins that are distinct from, but related to, the target molecule itself May modulate the activity of a protein unrelated to its intended target (never really know true effect of a drug to other parts of body and proteins, etc) The metabolic by products of the drug may be toxic ex. acetaminophen in liver can be processed through oxidation with cytochrome P450, further oxidized and the intermediate is highly reactive but doesn't matter because liver has high amounts of glucothione to conjugate the intermediate. Although if you overuse acetaminophen you will overuse glucothione so you will not be able to convert intermediate which is highly reactive, overdose

Protein Identification by Mass spectrometry

Measure the movement of ions (in gas phase, hard for proteins to be in that phase) in the electric/magnetic field in vacuum Mass/charge ratio m/z very accurate so can identify the proteins ESI (electrospray ionization) and SLD (soft laser desorption) treat the protein and make it ionized and make it go into gas phase 1. exercise the band and extract the protein 2. digest the protein of interest with trypsin 3. footprint of m/z vs abundance 4. protein sequence database searched for matches with theoretical masses calculated for all trypsin-released peptides 5. identification of corresponding gene

P450

Metabolizes potentially toxic foreign compounds without this enzyme, could lead to increase in unmetabolized, active drugs circulating and potentially accumulate in toxic levels in the blood

Overall structural organization of the core

N- terminal tail= largely unstructured, tails do not have well established secondary structures Histone fold= composed of 3 alpha helix joined by 2 loops. H2A and H2B form dimers. H3 and H4 can also form dimers

Z DNA

Narrowest Left handed helix Alternating anti and syn glycosidic bonds Based on crystal structure of CGCGCG May not be present in our bodies

Many different flavors of ubiquitination

Nearly every protein in body is regulated by ubiquitination, can form ubiquitane chain K48 chain= most common, leads to protein destruction by being recognized by Lys48

Two different ways to repair double strand breaks

Nonhomologous end joining homologous recombination Double strand breaks are very dangerous because once have break can be acted on by nuclease

Thumb of DNA polymerase

Not intimately involved in catalysis Interacts with DNA that has been most recently synthesized Serves to maintain the correct position of the primer and the active site Helps to maintain a strong association between the DNA polymers and its substrate (ensures DNA doesn't fall off)

DNA replication initiation in eukaryotes

ORC always bound to origin so NOT regulated, only time ORC lifts is when that piece of DNA needs to be replicated Pre-RC= ORC+ Cdc6+ Cdt1+ helicase Assembled during and only in the G1 phase Cdk (enzyme) phosphorylates proteins in Pre-RC complex. Phosphorylates Cdc6, then Cdc6 dissociates and helicase now active. ORC becomes phosphorylated Activate Pre-RC occurs only during S phase A pre-RC can initiate DNA synthesis only ONCE: being destroyed upon activation (don't want used more than once because genes could be replicated more than once leading to genome unstability)

Hexamer mechanism

Once you have enough nutrients hexameter can bind to origin, will recruit helicase helicase= DnaB in e.coli helicase carried by DnaC 1. Binding of DNA helicase to initiator protein 2. Loading of helicase onion DNA strand (helicase becomes activated) 3. Opening of DNA enables entry of RNA primase 4. RNA primer synthesis enables DNA polymerase to start first new DNA chain 5. Initiation of three additional DNA chains and formation of replication fork Two replication forks moving in opposite directions

DNA synthesis catalyzed by DNA polymerase

Only nucleotide with correct sequence can form H bond with Arg and Gln in palm region Riggers conformation change in finger region and Alpha helix closes down Then phsophodiester bond will form between primary strand and incoming nucleotide Phosphate will be released and hydrolyzed Leads to opening of DNA polymerase hand DNA will shift by one base pair and is ready to pick up next nucleotide 1. The correct positioning of an incoming dNTP causes the fingers to tighten, thereby initiating the nucleotide addition reaction 2. Nucleotide incorporation followed by pyrophosphate dissociation, which triggers the release of the fingers and translocation of the DNA by one nucleotide

Kinetics plays an important role in proofreading

Polymerization activity, if you have a mistake it will slow down the polymerization which leads to breakage of H bond between primary strand and template strand so you now have single stranded region which can migrate to exonuclease site. Once here can be hydrolyzed To reach proofreading site need to have a delay to allow uncoil from single stranded region

Development of drugs proceeds through several stages

Preclinical drug discovery Phase 1: Safety (no patient involved, only volunteers) Phase 2: Safety, efficacy, dosage (small number of patients) Phase 3: Safety, efficacy, and side effects (large number of patients, around 1000) Clinical use (monitor if safe and effective)

Comparison of replication fork in mammals and bacteria

Primase - associates with helicase in bacteria - associates with DNA polymerase in mammals Mammalian cells use two different DNA polymerases (alpha and S) Okazaki fragments are much shorter in mammalian cells Replication fork moves much slower in mammalian cells because deals with chromatin structure which slows down replication speed Alpha DNA polymerase in mammals synthesizes first 20 nucleotides of every Okazaki fragment then switches to DNA polymerase S (more processive and finishes up rest of Okazaki fragment)

DNA supercoiling problem

Problem when making DNA Induced by separating the strands of a helical structure If not solved it will block migration of DNA replication machinery

Ubiquitylation

Process by which one or more ubiquitin molecules are attached to a protein substrate molecule, which often results in the degradation of the tagged protein Ubiquitin with COO-(76 amino acid sequence, modifier, highly conserved in yeast and human sequence almost the same) can become activated by binding to lysine----> form isopeptide bond (amine bond, not a peptide bond)

Clone DNA

Process of producing replication of DNA within a host organism. Put target DNA into vector Target DNA fragment Cleave DNA with a restriction enzyme to yield compatible ends of vector and target DNA Purify DNA fragments and vector and ligate with DNA ligase Can be used to introduce in host of bacteria, can then use marker which is typically antibiotic resistance which leads to growth on plate

Restriction Endonuclease

Produced by bacteria to protect them from viruses by degrading incoming viral DNA Cut the DNA double helix at specific sites defined by the local nucleotide sequence Several hundred of them, each recognizes a distinct nucleotide sequence (each restriction endonuclease recognizes a different sequence) Fragments produced by restriction digestion can be easily joined together, ends as long as they are cut with same enzyme they can be cut and joined by ligase Palindromic= thats because each restriction endonuclease functions as a dimer so each subunit will recognize one strand. If palindromic it will look exactly same surface for subunit

Telomere Function

Protect eukaryotic chromosome Without telomeres, the ends of the chromosomes would be repaired, leading to chromosome fusion and massive genomic instability Solve the end replication problem (Requires telomerase) - active telomerase in cancer cells, stem cells and germ cells Telomeres are also thought to be the clock that regulates how many times an individual cell can divide. Telomeric sequence shorten each time the DNA replicates Once the telomere shrinks to a certain level, the cell can no longer divide. Its metabolism slows down, its ages and dies

Chromatin packing occurs at several levels

Proteins forming chromosome scaffold, each loop shows transcription unit -----> histone modifying enzymes chromatin remodeling complexes RNA polymerase----> gene can be expressed, uncoiling

Nucleotides Excision Repair

Pyrimidine dimer which is recognized by uVrA/uVrB protein complex which is important genes, for cells to be resistant to UV damage because UV damage can lead to pyrimidine dimer need protein to recognize and fix Nuclease (uVrC) cuts damaged strand, recruits nuclease to cut damaged strand, 8 nucleotides in front and 4-5 nucleotides after DNA helicase (uVrD) DNA polymerase and DNA ligase

Cas9 nuclease

RNA guided nuclease that is derived from bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Case (CRISPR-associated) system Using guide RNA to direct a nuclease

An individual X chromosome can be completely inactivated by heterochromatin formation

Random choosing to inactivate Xp or Xm don't know wy choose one over the other Condensation of a randomly selected X chromosome (either Xp or Xm) Direct inheritance of the pattern of chromosome condensation (1 to 2) Direct inheritance of the pattern of chromosome condensation (2 to 4) so ending with 4 only Xm active in this clone OR 4 only Xp active in this clone

Proofreading by DNA polymerase

Rare tautomeric form of C = C* happens to base pair with A instead of T Rapid tautomeric shift of C* to normal C destroys its base pairing with A Unpair 3'-OH end of primer blocks further elongation of primer strand by DNA polymerase 3'-to-5' exonuclease activity attached to DNA polymerase chews back to create a base paired 3'-OH on the primer strand DNA polymerase continues the process of adding nucleotides to the base-paired 3'-OH end of the primer strand (polymerization can continue)

RecA vs Rad51

RecA= prokaryotes Rad51= eukaryotes 1. RecA interweaves the DNA single strand and the DNA duplex in a sequence-independent manner (because just grabs DNA near by) 2. The DNA single strand searches the duplex for homologous sequence 3. Strand invasion: the single strand forms base-pairs with the complementary strand in the duplex (HETERODUPLEX)

Enzymes need for recombination

RecBCD= in bacteria which recognizes broken end/split. 3 subunits that forms complex Functions as helices and nuclease activity destroying resulting in single strand Once RecBCD reaches chi sequence, RecBCD only cuts from 5' end generating single strand region, leaving 3' in tact Generating single stranded region with 3' end which will function to invade adjacent DNA molecule

Movement of helicase along DNA double helix

Recruited to single strand DNA Moves and melts DNA helix by hydrolysis of ATP

Homologous recombination

Refers to recombination between two DNA molecules with extensive regions of sequence similarity (homology) Also called general recombination need to have that extensive similarity region

Palm DNA polymerase

Region composed of a beta sheet and contains the primary elements of the catalytic site Region binds two divalent metal ions that alter the chemical environment around the correctly base paired dNTP and the 3'-OH of the primer This region also monitors the base pairing of the most recently added nucleotides majority of catalytic residues bind here and metal ions

telomere

Repetitive DNA sequences at the ends of all eukaryotic chromosomes They contain thousands of repeats of the six-nucleotide sequence, TTAGGG (in human) or TTGGGG (in ciliate) Together with telomere binding proteins, they form a unique t-loop at the ends of each linear chromosome to protect the ends. The presence of t-loop protects the linear chromosome and prevents chromosome fusion Together with telomerase, telomere can help solve the end replication problem associated with the replication of linear DNA

Histone chaperones

Restore the full complement of histones to daughter molecules CAF-1= loading newly synthesized H3-H4 tetramer - can interact with sliding clamp so it knows that it is newly synthesized DNA - can interact with H3-H4 tetramer loading tetramer into newly synthesized DNA and starts formation of new nucleosome NAD-1 loading H2A-H2B dimer to fill gaps to fill complete histone octomer

Recombining DNA molecules by

Restriction digestion and enzyme mediated ligation (need restriction enzyme and ligases) 1. Cleave with EcoRI restriction enzyme 2. Anneal DNA fragments and rejoin with DNA ligase, Run under gel and purify. Put purify products together with ligase

Key techniques in recombinant DNA technology

Restriction-enzyme analysis Separating DNA fragments by gel-electrophoresis and purify easily Solid phase synthesis of nucleic acids Blotting techniques Polymerase chain reaction (PCR), amplify large amounts of DNA Rapid DNA sequencing

Topoisomerase I acts like a

Reversible endonuclease Energy stored in phosphodiester linkage between DNA and enzyme The original phosphodiester bond energy is stored in the phosphotyrosine linkage, making the reaction reversible. -OH group can attack phosphate group and can reform phosphiester linkage in DNA Spontaneous re-fromation of the phosphodiester bond regenerates both the DNA helix and the DNA topoisomerase Overall Topoisomerase I - make break - let it rotate - then leave

Sugar component in nucleotides

Ribose= RNA Deoxyribose= DNA

DNA replication occurs at __ phase of the eukaryotic cell cycle

S S (for synthesis)= the only period in the cell cycle for DNA synthesis All CORE histones (H2A, H2B, H3 and H4) are produced in S phase ONLY H1 is produced in G2 and G1 phase More than 50 copies of genes of histone cores, leading to mass production of transcripts early on in S phase. Transcripts are very unstable because not much of poly-A tail so quickly get destroyed at end of S-phase. Histone mRNA only present in S phase

Gene

Segment of DNA that contains the instructions for making a particular protein Some genes direct the production of an RNA molecule (like tRNA or microRNA genes) instead of a protein, as their final product

Drug candidates can be discovered by

Serendipitous observations (ex. penicillin and sildenafil) Screening libraries of compounds (ex. aspirin and lovastatin) Rational design based on 3-D structural information about their targets (ex. indinavir, rofecoxib, and an ideal opioid)

A DNA

Shorter and wider than B DNA Right handed alpha helix Dehydrated DNA anti glycosidic bond Double stranded RNA and DNA/RNA hybrid

Sildenafil

Sildenafil is very similar to cGMP so it is a mimic of cGMP increases smooth muscle relaxation Sildenafil can inhibit phosphodiesterase 5 leading to increase in cGMP = increase in muscle relaxation Ideal physiological response= increase smooth muscle relaxation for decreasing BP Side effect was the erection

Origin of replication in E.coli

Single origin which is very small has tandem array of 13-mer sequences (AT-rich), easier to open because only two hydrogen bonds so can easily open into single strand has binding sites for DnaA protein DnaA= first protein needed to initiate replication of E.coli

Origins of DNA replication on Ch3 of yeast

So many origins, not always all used in every cell cycle ORC binding site, unwinding region, Abf1 binding site (facilitates binding of ORC to ORC binding site) ORC= origin recognition complex In mammals, the binding sites for the ORC protein seem to be less specific, which leads to longer and less sharply defined sequences of replication origin

Winding Problem

Solves the winding problem/supercoiling that arises during DNA replication Need to rotated DNA in front of fork by one rotation but hard to do with long long strand of DNA so use enzyme to solve this problem = Type I DNA topoisomerase

Centromere

Specialized DNA sequence that allows one copy of each duplicated chromosome to be apportioned to each daughter cell

Chromatin

The complex of DNA and protein that makes up chromosomes

The double helix structure of DNA allows easy detection and efficient repair of lesions because

Structural alterations caused by lesions such as base mismatch and pyrimidine dimer can be easily DETECTED Unnatural bases generated by deamination can be easily detected (proteins that can take them out) It carries TWO separate copies of all the genetic information --- one in each of its two strands, thereby the backup copy can be used to restore the correct nucleotide sequence to the damaged strand - if one strand is broken, you can use other strand to correct - unless double strand break

TALEN

TALEs are composed fo tandem arrays of 33-35 amino acids repeats, each of which recognize a SINGLE BASE PAIR in the major groove if you know sequence easy to make fusion combination of tails to target nuclease to our specificities site= double strand break

Other forces that drive formation of fiber

Tails have basic residues (+ charge), can reach towards adjacent nucleosomes to form interactions Tails can interact with negative DNA or negative residues so nucleosomes come together

FISH Continued

The basic elements of FISH are a DNA probe and a target sequence. The probe is complementary to a region on the target Before hybridization, the DNA probe is labeled to contain a fluorophore The labeled probe and the target DNA are denatured Combining the denatured probe and target allows the annealing (forming a double stranded structure) of complementary DNA sequences)

Genome

The complete set of information in an organism's DNA Divided into chromosomes

Kd

The dissociation constant = measure of the strength of the interaction between the drug candidate and the target Example of ligand binding: R= receptor (drug target), L= ligand (drug candidate), RL= receptor-ligand complex R+L<---> RL Kd equals the concentration of free ligand at which one half of the binding sites are occupied Want lowered because that it is more potent meaning you need a lower drug concentration

Primer

The initial segment of a polymer that is to be extended on which elongation depends

Replication origin

The location at which duplication of the DNA begins Multiple origins, allows replication to occur at same time in those spots Typically have A-T rich so easy to open up

DNA synthesis at the leading and lagging strand are highly coordinated at the replication fork

The newly generated single stranded template for lagging strand synthesis is spooked out as a loop between the helicase and the DNA polymerase on lagging strand The size of the loop changes in the course of Okazaki fragment synthesis, much like a trombone Changes direction forming loop so both polymerases in same direction= synthesis becomes highly coordinated

Major and minor grooves in DNA double helix

The two glycosidic bonds are not diametrically opposite each other Each base pair has a larger side (major groove) and a smaller side (minor groove)

Topoisomerase II mechanism

Very active in actively dividing cells like in cancer cells. Topoisomerase II would be very active so if you can inhibit the enzyme using inhibitor (Etoposide) to block G- segment getting re-sealed so locks DNA in double strand break state which triggers apoptosis and cell death Etoposide (VP-16) inhibits/locks DNA in double stranded break state which can kills cells

Scientists manipulating genes

Way to harness the power of endogenous DNA repair pathway fro genome editing which is used by scientist to manipulate genes. Mutated gene and want to change with donor/replace with good gene inject donor DNA to the cell

Transcription-coupled repair

Way to prioritizes Mechanism used by cells to direct DNA repair (such as nucleotide exclusion repair), to the DNA sequences that are most urgently needed (i.e. those being actively transcribed when the damage occurs DNA damage causes RNA polymerase to stall at the site, and stalled RNA polymerase uses coupling proteins to direct the repair machinery to these sites Transcription coupled repair is especially advantageous in humans, because only a small fraction of human genome is transcribed at any given time. Thus important sequences can be repaired with the highest priority

Semi conservative replication of DNA

When parent DNA replicates in each daughter DNA, one strand serves as template and directs synthesis of new complimentary strand Template= a sequence of DNA or RNA that directs the synthesis of a complementary sequence

End replication problem

When replication reaches very end of linear chromosome when you are trying to synthesize lagging strand you have problem because not complete synthesis. last Okazaki fragment on lagging strand has RNA primer which will be removed. Need to copy information. Need to add primer somehow. If you don't do something each round of DNA replication will lose some DNA not getting replication. Telomere will shrink and shrink IF you can extend 3' end by more nucleotides can form primer base pair and complete copy of information DNA replication is bidirectional, polymerase moves 5' to 3', requires a labile primer

Pseudogenes (deeper explanation)

When reverse transcriptase is present in a cell, mRNA molecules can be copied into double stranded DNA In rare instances, these DNA molecules can integrate into the genome creating pseudogenes Because introns are rapidly removed from newly transcribed RNAs, these pseudogenes have the common characteristic of lacking introns. This distinguishes the pseudogene from the copy of the gene from which it was derived In addition, pseudogenes lack the appropriate promoter sequences to direct their transcription because these are not part of the mRNA from which they are derived

Chromatin domains within a chromosome

Will organized Each chromosome is comprised of many distinct chromatin domains= TADs (topologically associating domains), that are hundreds of Kb to several Mbs in length These chromatin domains are stable for many cell divisions, invariant across diverse cell types, and evolutionarily conserved in related species Because of the high degree of conservation, these chromatin domains have been considered the basic units of chromosome folding and regarded as an important secondary structure in chromosome organization

Homologous recombination :)

accurately repairs need sister chromatids to be close by, no deletion or insertion borrow information from sister homologous

Pseudogenes

arise from integration of reverse transcribed mRNA Nucleotide sequence that mimics the gene but are NOT expressed Chance event where cell infected by virus with reverse transcriptase and becomes DNA Reintegration is another chance event has no introns because spliced out in pseudogene =A nucleotide sequence of DNA closely resembling that of a functional gene but is not expressed

DNA replication is

bidirectional DNA polymerase reads the template strand in the 3' to 5' direction At replication origin often a lot of A and T's because easier to open since only 2 hydrogen bonds Creating replication bubble Replication fork= the junction between the newly separated template strands and the un-replicated duplex DNA Eukaryotic chromosomes contain multiple replication origins

N-glycosidic bond

between base and sugar (1 prime)

Inhibitors compete with substrates for

binding sites The concentration of the drug necessary to inhibit the enzyme effectively depends on the physiological concentration of the enzyme's normal substrate normal substrate with compete with inhibitor for binding site Benefit for noncompetitive inhibitor because you do not consider substrate concentration As substrate concentration increases, Kd increases

Nucleotides

building blocks for nucleic acids

Histone variants

can also alter nucleosome function, increases ways cells can regulate gene expression diversity histone variants can replace one of the four standard histones to form alternate nucleosomes Such nucleosomes may confer specialized functions to the nucleosomes into which they are incorporated H2A.X CENP-A (a H3 variant)

DNA primase

catalyze the formation of RNA primer

DNA synapsis

catalyzed by RecA protein DNA synapsis leads to base pairs forming between complementary strands from the two DNA molecules forming a HETERODUPLEX

Things that can harm DNA

cellular metabolism (metabolism generates reactive metabolites that can modify and damage it) viral infection radiation chemical exposure replication errors (DNA polymerase can make mistakes)

Histone binding domains are present in

chromatin remodeling and histone modifying complexes Domains guide proteins to correct space/place to modify them

Some DNA molecules are

circular and supercoiled Two forms: 1. Relaxed form of circular DNA, a circular DNA without any superhelical turns 2. Supercoiled form of circular DNA (more compact)

The specificity of replication is dictated by

correct base pairing H bonding to polymerase requires that a properly spaced base pair has formed in the active site Incoming nucleotide binds polymerase tighter if it contains the proper base Minor groove have H-bond acceptor in region can form H bond with Gln or Arg in palm region of DNA polymerase In palm region, Arg and Gln can check to make sure correct base pairing has occurred. Only when correct base pair, Arg and Gln can form H bond with it. Ensures finger region can change conformation

Chi

cross over hot spot instigator necessary for single strand invasion if dan molecule has lots of chi, it will easily undergo homologous recombination

Hypoxanthine

deamination of adenine Hypoxanthine looks a lot like guanine. Deamination fo adenine can generate hypoxanthine which can base pair with C instead of T so if you used as template, can lead to mutation

Sanger sequencing using

dideoxy chain termination No 3' -OH on sugar so terminates chain 1. Start with template of DNA, sequence you want to determine 2. Primer complimentary to sequence you want to determine 3. Polymerase reaction by DNA polymerase 4. One of the nucleotides in addition to normal form would have a small amount of dideoxy, if dideoxy incorporated the reaction would be terminated 5. Left with different length polymers of DNA terminating where dideoxy, indicating what the sequence was at that position. Do that for A, G, T, and C Paired with gel-electrophoresis allowing to separate DNA based on size - four reactions (one for dideoxy A, G C, and T) Gel is read 3' (TOP) to 5' (BOTTOM) or 5' (BOTTOM) to 3' (TOP)

Demethylase

direct repair Methylation Methyltransferase has a Cys residue which can recognize the error. Transfers methyl group to Cys side chain. Enzyme is now methylated and leaves Very expensive because methyltransferase can only fix one error can't can't be reused

Worst case

double strand break lead to chromosome fusion with all different segments after double strand break which leads to fusing= genome instability

FISH

fluorescence in SITU hybridization (SITU= in same place, identify location of gene while still on chromosome) Provides researchers with a way to visualize and map the genetic material in an individual's cells, including specific genes or portion of genes May be used for understanding a variety of chromosomal abnormalities and other genetic mutations

B DNA

fully hydrated DNA right handed alpha helix must cellular DNA anti glycosidic bond

Advantage of noncompetitive inhibitor as potential drug compared to competitive inhibitor

functions independently of substrate concentration

Comparison of the gene density in different organisms' genomes

gene density decreases as complexity increases Generally increase in genome size as complexity increases (sometimes not followed for plants because plants are not more complex than humans but have a larger genome size)

Unnatural bases

generated by the deamination of DNA nucleotides A wide variety of glycosylases have evolved to recognize different damaged bases - glycosylases are responsible for one type of unnatural base The DNA glycosylases travel along DNA using base flipping to evaluate the status of each base and remove the damaged base. Checks to see if base matches into active site of enzyme. If matches, wrong guy gets cleaved. Flip into active site to check

Rotation of a base about its glycosidic bond

greatly hindered, in most double helical nucleic acids, all bases are in anti-conformation syn= sugar on same side as base, base directly above sugar, may be found in ZDNA anti= base not directly above sugar

DnaB

helicase helicase (uVrD) Nuclease (uVrC)

Sliding clamp model

holds DNA polymerase on the DNA dimer larger hole so allowed to bind to DOUBLE strands sliding clamp increases processivity processive enzyme and distributive enzyme

Histone Code and Chromosome Structure

how histone modification could affect gene expression

If distance between polymerization site and proofreading site shortened

increase the fidelity (accuracy) of DNA replication

Certain types of chromatin structures can be

inherited when cells divide the structure can be directly passed down from a cell to its dependents Genetic vs epigenetic inheritance

Etoposide VP-16

inhibitor of Type 2 isomerase increases amount of double stranded break, so can use it to kill cancer cells

Base pairs on

inside, some exposed to major and others exposed to minor groove sites, helps proteins recognize identity of base pair Phosphate backbone on outside because charged and interact with aqueous environment Bases on the inside with non polar and hydrophobic

Ethidium

intercalating agent which can bind with DNA and slip into space between its base pairs DNA incorporated with many intercalating agents, DNA becomes elongated so if you use that DNA for replication, DNA polymerase becomes confused which leads to either insertion or deletion of some nucleotides leading to mutation

dideoxy method of DNA sequencing

is based on the termination of DNA synthesis after a dideoxy nucleotide gets incorporated and terminates extension of the DNA fragment. Dideoxyribonucleoside triphosphate (ddNTP) lacks a 3′ "OH" group, which terminates DNA synthesis and prevents strand extension at 3' end Method used to deduce DNA sequence

Artenisnin

isolated from plant the most effective treatment now available against malaria

PHD

it takes PHD to read some marks on a nucleosome PHD= plant homeodomain, binding pocket specific for trimethylated part Trimethylate acts as a mark to recruit proteins with PHD domain to region of chromatin One of many PHD domains that recognize methylated lysine on histones Forms new binding site allowing PHD to bind to tail specifically to histone tail binding highly increases

Nucleic acids formation by

joining nucleotides together through a phosphodiester linkage OH from sugar 1 acts as a nucleophile and attacks phosphate on sugar 2 Chain has directionality - beginning with 5' end of chain with phosphate group - end with 3' end of chain with sugar

Sliding clamp

keep DNA polymerase firmly on the DNA

SSB

keep single stranded DNA in an extended, relatively inflexible conformation

DNA polymerase holoenzyme

large enzyme complex at the replication fork

The targets of drugs in current use

largest protein family for drug target = GPCR enzymes nuclear receptors voltage gated ion channels ligand gated channels solute carriers etc

Zigzag

linker DNA needs to be long because has to cross axis

DNA ligase model

links two fragments by forming a phosphodiester bond (look at class notes for model) Adenylation= type of activation adding AMP unit to atom so becomes activated

Clamp loader

load sliding clamp to DNA

MutL

looking for GATC (methylated in parents and unmethylated in new) that is close by to distinguish the newly synthesized strand from the template strand (looking)

SSB helps to

maintain the structure of single stranded DNA (PROTECTION) DNA needs to be protected. It needs to be protected because 1. Some regions of DNA could base pair with each other (hairpin) which blocks movement of DNA polymerase so that's not good 2. easily attacked by hydrolytic attack so easier to be degraded SSB is highly cooperative, when one molecule binds it makes it easier for the next molecule to do so. Each SSB has 2 binding sites. 1= DNA 2= SSB, SSB can bind to itself=cooperative and rapid

cDNA synthesis

making a complementary DNA sequence of your RNA with reverse transcriptase 1. Start with mRNA (template) 2. Hybridize with poly t primer 3. Make complementary DNA copy with reverse transcriptase (DNA polymerase synthesizes DNA copy composition to mRNA) 4. Degrade RNA with RNaseH 5. Synthesize a second cDNA strand using DNA polymerase 6. Synthesize a second cDNA strand using DNA polymerase; RNA fragment acts as primer. Put cDNA into library to identify gene in tissue

DNA polymerase utilizes

metal ion catalysis 2 metal ions in each active site Mg2+ will bind to primary strand to activate a 3' -OH group making better nucleophile Second Mg2+ will bind to incoming nucleotide and help to position it

Types of DNA damage

mis-incorporation of a single base (replication error, wrong nucleotide) chemical modification of bases chemical cross-links between the two strands of the double helix breaks in one or both of the phosphodiester backbones (single or double strand break) Thousands of random changes occur in DNA of each human cell every day

Without repair, spontaneous changes in DNA can produce

mutations, like deamination or depurination short period of time to fix mutation before it becomes permanent, repair prior to replication if you don't fix, then you let if become template for replication leading to mutation, damage strand as template (leading to mutation)

Stability of the polynucleotide backbone

negative phosphate can repel nucleophile making more stable. In presence of -OH can activate adjacent OH leading to attack of adjacent phosphodiester linkages so can break= so RNA less stable - RNA is less stable than DNA For DNA, sugar phosphate backbone makes it really stable. Negative on phosphate can repeal nucleophile= more stable Exonuclease= removes nucleotides from end to end one of a time in DNA 5'- exonuclease 3'-exonuclease Endonuclease: speed up cleavage of internal phosphodiester linkages

Why DNA synthesis occurs 5' to 3' Direction

new nucleotide always added to 3' end. If added to 5' end it is not compatible with proofreading

Mismatch in eukaryotes

nick shows you new strand due to Okazaki fragments before joined together don't use MutH because already have nick

MutH

nicks the unmethylated GATC sequences (bacteria only) makes cut on damaged sequence (hydrolyzing)

Genome editing with programmable nucleases

nuclease targets specific locus and break double bond Zinc-finger nucleases (ZFN) Transcription activator-like effector nucleases (TALEN) Cas9 nuclease

Base, nucleoside, nucleotide

nucleoside= base+sugar (connected by glycosidic bond) nucleotide= base+ sugar+ phosphate Adenine (base), adenosine (nucleoside), adenylate (nucleotide) Guanine, guanosine, guanylate cytosine, cytidine, cytidylate uracil, uridine, uridylate thymine, thymidine, thymidylate AMP: adenosine monophosphate ADP: adenosine diphosphate ATP: adenosine triphosphate dAMP: deoxyadenosine monophosphate dADP: deoxyadenosine diphosphate dATP: deoxyadenosine triphosphate

Structural organization of the nucleosome

nucleosome includes around 200 nucleotide pairs of DNA (ex. DNA with 1000 base pairs can form 5 nucleosomes) Beads on a string form chromatin, connected by linker DNA Linker DNA digested by nuclease which removes linker DNA Released nucleosome core particle can be dissociated with high concentration of salt = H2A, H2B, H3, and H4

Two metal ions bound to DNA polymerase catalyze

nucleotide action The two metal ions are held in place by interactions with two highly conserved aspartate residues Metal ion A= primarily interacts with the 3'-OH, resulting in reduced association between the O and the H. Produces a powerful nucleophile Metal ion B= interacts with the incoming dNTP to position it for nucleophilic attack and to stabilize the resulting -PP (pyrophosphate) - makes sure phosphate atom and oxygen are perfectly align

Albumin

once in bloodstream some of drug tends to be hydrophobic so tend to aggregate so utilize albumin to get into right compartment carrier protein Human serum albumin (many hydrophobic pockets which drugs can go into, "taxi") serves as the carrier for hydrophobic compounds for their distribution Tend to aggregate so albumin has hydrophobic pocket and can then distribute to other parts of the body can have drug drug interaction in albumin because it has many pockets so need to be careful when taking different medications Can visualize the distribution of the drug fluconazole by PET scan

Phosphate component of nucleotides

one phosphate group (ester linkage) as in AMP two phosphate groups (anhydride linkage) as in ADP three phosphate groups as in ATP

DNA helicases

open up the DNA helix

Vector normally used

plasmid Should have... 1. ori (origin of replication: site where DNA replication can start) 2. marker gene (allows cells to grow when exposed to certain conditions. Allows you to identify the host that contains the plasmid) 3. Polylinker (region with multiple restriction enzyme sites for insertion of DNA fragment)

X chromosome inactivation

process that occurs in female mammals in which one of the X chromosomes is randomly turned off in each cell XIC becomes activated can be transcribed generating RNA= XIST. XIST ends product of XIC locus. XIST= noncoding RNA bind to same X chromosome that produced it and can recruit proteins to condense x-chromosome XIST= X-inactivation specific transcription, noncoding RNA so NOT a PROTEIN Dosage compensation for females= only one active X chromosome and one completely inactivated if had both X chromosomes that would be too large

The telomerase

protein RNA complex that carries an RNA template. RNA region in active site that has sequence complementary to telomere repeat so function as template two direct DNA synthesis Can solve end replication problems Reverse transcriptase because use RNA as template for synthesis of DNA to be able to extend 3' end

encountering with reactive metabolites

reactive oxygen species (H2O2, O2-) react with DNA and damage

Distributive enzyme

releases its polymeric substrate between successive catalytic steps not very efficient

DNA topoisomerases

relieve helical winding and DNA tangling = DNA topoisomerase I: single strand break (solve winding problem) = DNA topoisomerase II: double strand break (solve tangling problem)

RNase H

remove RNA primer Recognizes RNA-DNA hybrid

Factors that can cause accidental lesions in DNA

replication errors spontaneous loss of purines and deamination fo certain bases encountering with reactive metabolites radiation exposure to substances in the environment

DNA ligase

seal together the Okazaki fragments

A code-reader-writer complex can

spread chromatin changes along a chromosome Gene regulatory protein binds to unique chromatin region. Protein can regulate specific gene. Can recruit histone modifying enzymes which can function as a reader and modify histone tails which willl write the code. Code can be read by reader protein. Reader protein carry their own writer so form reader writer complex. Writer carried by reader can write the exact same code as code recognized by the reader so writer will write the code for adjacent nucleosome and recognized by next reader writer complex. Code can be spread. Gene typically very long so can spread but eventually needs to stop otherwise the same code throughout

Human genome sequence

the complete nucleotide sequence of DNA in the 24 nuclear chromosomes and the mitochondria DNA length = 3.2 x 10^9 nucleotides Number of genes = approximately 25,000 Percentage of DNA sequence in exons (coding regions) = 1.5 % majority= noncoding intron regions

RecA

type of single strand binding protein different from SSB because has multiple DNA binding sites so is able to bind to single and double strand and form 3 stranded structure highly cooperative

replication errors

uncorrected misincorporation resulting in mismatched base pairs need to correct errors prior to replication

Topoisomerase II

untangles DNA has 2 subunits, identical and 2 domains on each subunit One subunit with ATP binding sites Other subunit with Try, so Tyr residues can bind to DNA and will each break one of the strands making double strand break creating a gate

FISH analysis

using a different mixture of fluorochromes for marking the DNA of each chromosome, detected with seven color channels in a fluorescence microscope, allows each chromosome to be distinguished in 3D reconstructions

DNA polymerase

very accurate, has to pick up correct nucleotide and incorporate it into primary strand. Can check to make sure correct nucleotide includes exonuclease which acts as deleting key

Negative supercoiling

very common because need to open double helix thermophile= positive supercoil, higher temperature to uncoil. Increases stability of double helix structure

Histone methyl transferase

very specific

spontaneous loss of purines and deamination of certain bases

water present and leads to loss of purines and some amino groups in certain bases spontaneous hydrolytic attack