BioChem Final Exam- New Material

introns

sequence of DNA that is not involved in coding for a protein --> in eukaryotic genes - get spliced out to link exons together

Okazaki fragments

small fragments of DNA produced on the lagging strand during DNA replication, joined later by DNA ligase to form a complete/continuous strand

How do replication and transcription affect DNA supercoiling?

some of the energy required for unwinding comes from taking advantage of the supercoiling - topoisomerases

How did the results of the Meselson and Stahl experiment rule out the dispersive model of DNA replication: where parent DNA strands are cleaved into pieces of random size, then joined with pieces of newly replicated DNA to yield daughter pieces?

- If random, dispersive replication takes place, the density of the first gen DNA in the Meselson-Stahl experiment would be the same as was actually observes: a single band midway between heavy and light DNA - HOWEVER, in 2nd gen, all the DNA would again have the same density and would appear as a single band, midway between the band of first gen and that of light DNA --> ***this was not observed - Two bands were obtained in the first experiment, ruling out dispersive model

DNA of phage M13 is A:23%, T:36%, G:21%, C:20%

- In the two strands of a duplex DNA hargaff;s rule = sum of pyrimidine nucleoides equals that of purines in DNAs so A = T, G = C, and A + G = C + T - in this DNA, A does not = T, and A + G does not equal C + T --> lack of equality between purines and pyrimidines tells us that it is NOT double-stranded but single-stranded; expected relationships not seen - M13 DNA is double-stranded only when replicating in the host cell

Prokaryotic topoisomerase II is inhibited by antibiotic which binds to B subunit to inhibit bacterial DNA replication --> topo II do and why would this be an effective antibiotic?

- Topo II decreases the linking number by cleaving the DNA duplex, allowing another section of the bacteria's circular DNA duplex to pass through the break - inhibition prevents the addition of negative supercoils necessary for strand separation during replication **no induction of underwinding

Thick bands in gel are DNA:treated chromatin with enzyme that degrades DNA, then removing all protein and subjecting purified DNA to electrophoresis. Numbers = the position to which a linear DNA of indicated size would migrate: - What does gel say about chromatin structure? - Why are bands thick and spread out rather than sharply defined?

- bands have periodicity of 200 bp (200 bp intervals) --> chromatin is protected from nuclease digestion at regular intervals of 200 bp ***suggests the nucleosomal cores (146 bp) provide the protection --> nucleosomes, thus, are in a fairly regular array, occurring about once every 200 bp - nuclease cuts the regions of double-stranded DNA that link but do not bind to the nucleosom cores- specifically the spacer regions of about 60 bp; these regions are not always digested to completion --> some bands then correspond to the DNA from single nucleosomes (200 bp), others to two nucleosomes (400 bp), etc. ***if the nucleosomes were randomly distributed in the chromatin, a large number of differently sized DNA fragments would be generated by nuclease cleavage; in this case, a heterogeneous population of DNA fragments would have smeared through the gel - bands are thick because the spacer is fairly long (60 bp) relative to the nucleosomal core (146 bp) --> the nuclease can cut essentially anywhere in the spacer, so the band corresponding to, for example, mononucleosomes has DNAs ranging in length from 146 to 206 bp

Beadle an Tatum's work with Neurospora crassa

- genes, messages, and proteins are COLINEAR - gave way to one gene-one enzyme (polypeptide) - alternative splicing gives different versions of proteins

structure of DNA

- made up of deoxyribonucleotides linked 5' to 3' - double helix (2 strands) - helix is held together by H-bonds - the 2 strands are antiparallel to one another (run in opposite directions) - sequence of bases in the strands are nonrandom ***C-G = 3 bonds ***A-T = 2 bonds

Switch 15NH4Cl to 14NH4Cl for three generations (an eight fold increase in population): What is the molar ratio of hybrid DNA to light DNA?

- semiconservative replication --> after 3 generations the molar ratio is 2 / 6 --> 0.33

E. coli (and eukaryote?) DNA polymerases

1. DNA Pol III: principal one for replication 2. DNA Pol II 3. DNA Pol I: important for lagging strand synthesis (Kornberg worked on it)

eukaryotic DNA polymerases

1. DNA Pol a 2. DNA Pol delta (has 3' to 5' exonuclease activity) 3. DNA Pol epsilon (has 3' to 5' exonuclease activity)

enzymes involved in DNA replication

1. DNA polymerase 2. helicase 3. topoisomerase 4. DNA binding proteins (i.e. SSB) 5. primases 6. ligases ***all together: replisome

requirements for DNA replication

1. Mg2+ cofactor 2. template strand 3. primer: a 3' OH group to attach the next base 4. high processivity (kind of)

phosphates of DNA in replication

1. a-phosphate: stays 2. B and y get cleaved

ligase mechanism

1. adenylylation of DNA ligase: add AMP group (adenine) 2. activation of 5' phosphate in nick 3. displacement of AMP seals nick: pushing it out as a leaving group - by DNA ligase ***gives one continuous strand

DNA polymerase fixing mechanisms

1. evaluation of the base pairing (incorrect ones can be put in through tautomerization) 2. proofreading activity (3' --> 5' exonuclease)

types of DNA recombination

1. homologous recombination 2. site-specific recombination

steps of each central dogma process

1. initiation: how it starts 2. elongation: how it continues 3. termination: how it stops

homologous recombination

1. repairs lesions in DNA 2. eukaryotes: transient physical link between chromatids to keep chromosomes together at the first meiotic cell division 3. enhances genetic diversity crossing over between sister chromatids varies alleles across chromosomes --> genetic diversity ***occurs between sister chromatids during meiosis leading to genetic diversity - end up with haploid gametes that may/may not have same order of alleles as the parents

qualities of DNA replication

1. semi-conservative 2. bidirectional ***has to be accurate

elements of noncoding regions (linear [eukaryotic] chromosomes)

1. telomeres 2. centromeres 3. simple repeated sequences telomere, unique sequences (genes), dispersed repeats between telomere and centromere and centromere and telomere, then telomere at other end

Which direction does DNA replication proceed in?

5' --> 3' direction ***which means you ADD at the 3' end which becomes an issue b/c bidirectional

Which direction are nucleotides linked?

5' to 3'

central dogma

DNA (replication) --> RNA (transcription)--> protein (translation)

single strand binding proteins (SSB)

DNA binding proteins: binds to single-stranded DNA to avoid DNA rewinding back; covers up naked DNA (protects it)

structure of chromosomes

DNA is wrapped around nucleosomes = chromatin ***extent of how tightly wound/packed DNA is in histones determines gene expression

processivity

DNA polymerase must have high processivity: incorporate many bases before falling off the template (basically just being efficient) - doesn't stay at the fork all the time, jumps on and off but high processivity means mostly on

accuracy of DNA replication

DNA polymerases have to deal with 4 different deoxynucleotides as substrates: looking for the correct base pairing in case it must be fixed

steps of central dogma

DNA replication --> transcription --> translation

hemimethylated DNA

DNA sequence with one methylated strand (template) and one unmethylated (new) strand: in this state you realize the mutation because the cell recognizes the old strand is correct and has to make a change in the new strand

elongation of replication in E. coli

DNA synthesis (mostly DNA Pol III): 1. continuous synthesis on the leading strand proceeds as DNA is unwound by the DnaB helicase - clamp-loading complex? 2. okazaki fragment synthesis nears completion; primase binds to DnaB, synthesizes a new primer, then dissociates 3. a new B clamp is loaded onto the new template primer by the clamp loader; synthesis of a new okazaki fragment is completed on the lagging strand 4. lagging strand core subunits are transferred to the new template primer and its B clamp, and the old B clamp is left behind 5. the next B clamp is readied as okazaki fragment synthesis is initiated green RNA needs clip out: done by DNA Pol I because it has 5' to 3' exonuclease activity and can fill in where the RNA primer used to be ***THEN ligase activity is necessary to link the new pieces of DNA together into a continuous strand - requires energy

mechanism common to all DNA polymerases

DNAn + dNTP --> DNAn+1 + PPi 1. add a nucleotide to the end 2. release pyrophosphate to provide energy: because you can't go back here, this step of PPi release pulls the entire reaction to the right

Gene has 21,000 bases but weight is 110000 --> prokaryote or eukaryote?

Gene is 7x as long as it needs to be for the 1000 amino acids that make up the protein --> presence of untranslated regions of DNA within the coding sequence indicates introns, so eukaryote

Describe the organization of human chromosomes.

Genes for histones and ribosomal RNAs are structural genes ***NOT all the genetic info of the cell is encoded in the nuclear, chromosomal DNA

origin of replication in E. coli

OriC

methylation of histones

K and R residues: often associated with heterochromatin/euchromatin switch

acetylation of histones

K residues: usually associated transcriptional activation

Are there protease-sensitive regions of DNA between the nucleosomes?

No

Do histones grip the DNA double helix like a fist, with the DNA in the center?

No

Do the amino acid sequences of histones vary from species to species?

No

Does DNA constitute 90% of the mass of chromatin and the rest is protein and a small amount of RNA?

No

Is formation of the nucleosome structure the only condensing step in chromosomal DNA?

No

phosphorylation of histones

S and T residues: usually associated with chromatin condensation during mitosis and some transcriptional activation

Are histones basic proteins that make up about half the mass of chromatin?

Yes

Does the nucleosome structure act to condense DNA by decreasing its length?

Yes

topoisomerases

enzymes that change/can introduce supercoils - Type 1 - Type 2 ***some drugs inhibit transcription/regulate via these by inhibiting winding/unwinding DNA

DNA primer

a 3' OH group to attach the next base: 3' OH uses nucleophilic attack to release PPi and attach an a-phosphate (meanwhile B and y get hydrolyzed)

replication fork

a Y-shaped point that results when the two strands of a DNA double helix separate so that the DNA molecule can be replicated

lagging strand

a discontinuously synthesized DNA strand that elongates by means of Okazaki fragments, each synthesized in a 5' to 3' direction away from the replication fork; have to add at the replication fork because DNA polymerase won't add to the 5' end - then has to back up and keep going: you end up with Okazaki fragments because of the backing up

nucleotide-excision repair

a pathway to remove bulky DNA lesions (when you have bulky add-ons to DNA) i.e. chemical stuck on base, something through UV light? 1. two clips on either slid of the mutation (excinuclease) 2. DNA helicase takes out chunk 3. DNA Pol I in E. coli and epsilon in humans adds on to the OH in nick to add good stuff back after chopping out the bad part 4. DNA ligase repairs second nick ***large distortions in the helical structure of DNA require this, which uses unusual enzyme ABC exinuclease, which makes 2 cuts (not 1) in the DNA in order to remove the damaged portion

direct repair

accomplished by an enzyme called photolyase 1. a blue-light photon is absorbed by MTHF-polyGlu 2. the excitation energy passes to FADH- in the active site 3. the excited FADH- donates an electron to the pyrimidine dimer to generate an unstable dimer radical 4. electron rearrangement restores monomeric pyrimidines 5. electron is transferred back to the flavin radical to regenerate FADH- UV light comes in, often with TT dimers (pyrimidine dimers) --> aducts get created in the presence of UV light and you need to get rid of them: uses FAD cofactor to get rid of the covalent bond ***unrepaired damage gets "fixed" in the genome = a lasting mutation ***does not involve extraction of a base or nucleotide and is thus limited to those types of damage that can be fixed by removing or reversing the damage-- usually a methyl group or extra bonds between pyrimidines

proofreading activity of DNA polymerase

all DNA polymerases have 3' --> 5' exonuclease activity: can look at the last base and tell if it's correct - if incorrect, ships it out for 3' to 5' exonucleaseactivity to chop out the last base pair ***instead of letting the mistake get incorporated into the next strand, chop it out and try again 1. polymerase mispairs dC with dT 2. polymerase repositions the mispaired 3' terminus into the 3' --> 5' exonuclease site 3. exonuclease hydrolyzes the mispaired dC 4. the 3' terminus repositions back to the polymerase site 5. polymerase incorporates the correct nucleotide, dA

types of DNA repair mechanisms in E. coli

all in moderate mutagenic conditions: 1. mismatch repair 2. base exclusion repair 3. nucleotide excision repair 4. direct repair 5. error-prone repair ***unrepaired DNA damage gets "fixed" in the genome = a lasting mutation - usually a change in the nucleotide sequence, in protein coding sequence, therefore protein doesn't work - this is also necessary for evolution though

DNA polymerase a

changes RNA --> DNA primers - no 3' to 5' exonuclease function

plasmid

closed circular DNA molecules

Type 2 topoisomerase

cuts both strands of DNA; catalyzes double strand breaks- changes the Lk in increments of 2

site-specific recombination

depending on the location and orientation of the sites of recombination, the outcome varies - can invert things (turn around?) - can delete or insert

Watson & Crick hypothesis for DNA replication

each strand serves as the template for a new strand; DNA replication is semi-conservative

one gene-one enzyme theory (polypeptide)

genes act through the production of enzymes, with each gene responsible for producing a single enzyme that in turn affects a single step in a metabolic pathway ***connects genotype (genetic composition) to phenotype (visual trait) order of bases in DNA reflects order of bases in RNA --> translates to protein ***now we know alternative splicing gives different versions of proteins

chromatin

granular material visible within the nucleus; consists of DNA tightly coiled around proteins - heterochromatin: tightly wound - euchromatin: less tightly wound --> ***genes more highly expressed because enzymes have access to them

E. coli genome

has many genes to regulate DNA metabolism - all required for replication and repair --> because lots of genes, these processes happen at high fidelity (need to do things correctly, although you need a little mutation not extensive enough that life isn't compatible (why there are repair systems in place))

supercoiling topology

how wound up DNA is --> form of potential energy

relationship between number of amino acid residues in the enzyme and the number of nucleotide pairs in its gene

i.e. 192 amino acid residues in protein but gene has 1440 base pairs - each amino acid = coded for by 3 base pairs --> 576 base pairs are coding - additional base pairs could be introns (noncoding DNA, interrupting a coding segment) or could code for a signal sequence (or leader peptide) - eukaryotic mRNAs also have untranslated segments before and after the region for the polypeptide chain, which also contribute to the extra size of genes

DNA Pol III

in E. coli: principal one for replication --> complex that has proofreading ability (but no 5' to 3' exonuclease activity from 5' end) - has many subunits ***structure: holoenzyme clamp, then DNA goes into the donut hole

semi-conservative replication

in each new DNA double helix, one strand is from the original molecule, and one strand is new --> each strand serves as template for new strand

ligase

link pieces (fragments) of DNA together

initiation of replication in E. coli

lots of proteins are required: 1. DnaA protein binds to oriC origin to help unwind DNA at 13 bp sites that are A/T rich 2. DnaB (helicase) comes in, helps unwind more 3. also need DnaC and others i.e. primase to lay down RNA primer, SSB, topoisomerase 1. DnaA gets loaded onto the binding sites, then with HU (a histone-like protein) and ATP, unwinds the 13 bp repeats so DnaB can be loaded along with DnaC ***requires hydrolysis of ATP for energy 2. lagging strand: start laying down --> first primer, then DNA polymerase extends this to make DNA, then backs up, adds new primer, continues, backs up, new primer, etc.

primase

make a short RNA primer to get things going (primer then provides 3' OH)

Type 1 topoisomerase

makes a nick in one DNA strand to relieve supercoiling; catalyzes single strand breaks with strand passing over- changes the Lk in increments of 1 - break one strand at a time, one strand can be unwound around the other

Dam methylase

methylates adenines in GATC sequences of E. coli; puts methyl groups on the new strand

DNA replication in E. coli vs. eukaryotes

more complicated in eukaryotes 1. E coli = one circular chromosome and you get cell division after new circle separates - linear chromosomes present problems for eukaryotes: how to replicate the ends? 2. E coli = one origin of replication (oriC) - multiple origins of replication: autonomously replicating sequences (ARS) are analagous to OriC 3. several DNA polymerases with different functions in eukaryotes: 1. DNA Pol a 2. DNA Pol delta (has 3' to 5' exonuclease activity) 3. DNA Pol epsilon (has 3' to 5' exonuclease activity)

heterochromatin

more tightly wound, less available for transcription

simple repeated sequences

scattered throughout- function unknown

underwound DNA

most DNA in cells: promotes strand separation but "strains" the DNA molecule --> supercoiling reduces the strain and leaves the DNA in more accessible, underwound form --> because supercoiling is energetically more favorable than actual strand separation, which requires breaking hydrogen bonds between base pairs - supercoiling controlled by topoisomerases

DNA polymerase delta

mostly for lagging strand synthesis - has 3' to 5' exonuclease activity

DNA polymerase epsilon

mostly for leading strand synthesis - has 3' to 5' exonuclease activity

purpose of Mg2+ cofactor in DNA replication

neutralizes the negative charges from phosphates in the template

linking number (Lk)

number of times DNA winds in right handed helical direction when constrained to a single plane; the number of times the strands cross each other - number of supercoils defined mathematically i.e. if relaxed DNA has 200 Lk, then 2 positive supercoils Lk = 202, 2 negative supercoils Lk = 198

error-prone repair

occurs when DNA replication gets stalled; a lot of mistakes happen in the process ! - involves DNA Pol II ***SOS response: cells try to recombine DNA in a way compatible with life unrepaired lesion --> single stranded DNA --> recombinational DNA repair or error-prone repair OR unrepaired break --> double-stranded break --> recombinational repair ***extreme cases of extensive damage: allows replication even without a template, which increases mutation rates - risky but preferable to an inability to replicate at all

euchromatin

open structure, more available for transcription

oriC

origin of replication in E. coli: provides a binding site for important proteins in the R regions --> protein A will come unwind to get DNA started R regions: DnaA protein binds here, helps unwind DNA at 13 bp sites - A/T rich regions for unwinding because only 2 and not 3 base pairs (makes it easier to unwind) - DnaA recognizes the origin sequence of blue boxes to unwind!!! DnaB moreso with helicases

epigenetics

post-translational modifications of histones regulates DNA packaging

histone

protein molecule around which DNA is tightly coiled in chromatin - special ones for centromeres, tetromeres, etc. ***DNA is negatively charged, so chromatin is positively charged to associate with it ***can be modified (co- and post-translational modifications like phosphorylation) but here histones regulate the winding and unwinding histones have tails that can be modified by methyl groups, acetyl groups that determines how tightly DNA is wound ***they make DNA NEGATIVELY supercoiled (changes Lk), can open up to get proteins in there and use energy of unwinding for other things

Meselson & Stahl with DNA replication

provided experimental evidence that DNA replication is semi-conservative: 1. grew E. coli in heavy nitrogen and spun it so that where it rests - original parent molecule had lowest band (blue) with heavy DNA - first-generation daughter molecule had medium band (purple) with hybrid DNA --> one strand blue, one strand red ***showed each blue strand served as template for red - second-generation daughter molecules had high band of light DNA (red) and medium (purple) hybrid DNA ***showed fully red strands because newly replicated strand in the first generation acted as a template over time the ratio went from 1:1 to more light like 10:1 ***and you never get back blue band of heavy DNA

topoisomerase

relieves positive supercoils

telomeres

repeated DNA sequences at the ends of eukaryotic chromosomes; for maintenance of ends of linear chromosomes (more in RNA metabolism) --> caps on the end

bidirectional replication

replication starts at the origins of replication - in E. coli: OriC is only origin: replication proceeds at two replication forks at the same time --> duplex opens up and at each fork you get replication happening ***ultimately two new daughter duplexes that are both circular this becomes an issue for leading/lagging strand

termination of replication in E. coli

requires separation of DNA circles (activity provided by topoisomerase); had a mess of DNA with chromosomes stuck together and need topoisomerase to unlink them terminator slowing things down, but when we get to this point tells us to separate

How do incorrect base pairs get incorporated?

tautomerization: appears to have base pair property different from the normal form

purpose of template strand in DNA replication

tells DNA polymerase which bases to add

Ames Test

test in which special strains of bacteria are used to evaluate the potential of chemicals to cause cancer: how mutagenic is a carcinogen? --> measuring the mutation rate, trying to understand how many mutations can happen (max) so that life is still compatible 1. dropped drug into the middle of the plate: different concentrations in each plate, but drug seaps out from the center - first plate: not a lot of colonies coming up, close to dot --> for background rate of mutation - second plate: drug seaping into the agar on a concentration gradient but once at a concentration with just a few mutations, we see colonies again - third plate: less drug is seaping in - fourth plate: only a little bit of drug ***between 2nd, 3rd, and 4th plates, 2nd has the biggest zone of inhibition --> drug concentration is so high that bacteria are mutating so much it's incompatible with life (cells = dead) ***largest zone = least compatible with life

mismatch repair

the cell determines which strands are templates because they are methylated- if there are mismatches, then the cell "assumes" the unmethylated (new) strand needs to be fixed 1. for a short period following replication, the template strand is methylated and the new strand is not (hemimethylated DNA) 2. after a few minutes the new strand is methylated and the two strands can no longer be distinguished (by Dam methylase- puts methyl groups on the new strand) - at GATC sequences 3. the Mut proteins, MutL and MutS, recognize the damage and cleave the DNA (in an ATP-dependent manner) so that repair enzymes can bind and repair the mismatch from the nic ***repairing the damage requires a number of different enzymes puts methyl groups on A residues --> in the process, theres a time where A residues don't get methylated right off, and in this time you can distinguish between the old strand and new/unmethylated strand then mismatch enzymes bind/repair the mismatch from the nick (chop out the bad part, fill in with DNA Pol I activity) --> nick in the unmethylated/new strand that you fill in with good stuff --> get loaded onto mismatches (recognized by MUT proteins) using ATP energy to chop out/make a single stranded cut in the new/unmethylated strand ***finds and replaces incorrectly matched (i.e. wrongly base-paired) bases after replication --> discrimination between parental and daughter strands is achieved through methylation of the parental strand

DNA supercoiling

the formation of additional coils in the structure of DNA due to twisting forces on the molecule; DNA exists as a coil, much like a phone cord ***results in the compaction of DNA structure ***supercoiling topology (how wound up it is) governs many reactions and is a form of potential energy - plasmid DNA can be relaxed or supercoiled positive supercoiling: right handed direction? (left strand over right) --> must be unwound negative supercoiling: left handed direction? (right strand over left) ***you wind up chromosomes in such a way to help get proteins in there, push DNA or RNA polymerase through the gaps

nucleosome

the functional unit of chromatin, a "bead": octamer containing two of each histone protein H2A, H2B, H3, and H4 - H1 binds to the linker region (the regions between the histones/histone cores) ***the DNA coiled around histone = nucleosome ***nucleosomes regulate gene expression and DNA compaction

leading strand

the new complementary DNA strand synthesized continuously along the template strand ***toward the replication fork in the mandatory 5' to 3' direction - this is the way that DNA polymerase adds (to the 3' end)

centromeres

the point on a chromosome by which it is attached to a spindle fiber during cell division; for attachment of the mitotic spindle, essential for mitosis --> where the spindle attaches, without it the chromosomes get lost

tautomerization

the rearrangement of bonds in a compound, usually by moving a hydrogen and forming a double bond

base excision repair

two enzymes remove the damaged base, then DNA Pol I comes in to fill the gap, and ligase repairs the nick 1. DNA glycosylase takes out damaged base 2. AP endonuclease creates nick 3. DNA Pol I brings in new NTP 4. DNA ligase patches up nick base is gone, nick happens, DNA polymerase comes in and repairs, ligase patches up --> chopping base not nucleotide ***recognizes lesions such as those produced by deamination of purines and pyrimidines; it first removes the base itself, then the rest of the nucleotide, and finally refills the gap using DNA Pol I

helicase

unwinds the DNA duplex: requires ATP