Kaplan Biochemistry Ch. 6

PCR

(polymerase chain reaction) a method used to rapidly make multiple copies of a specific segment of DNA; can be used to make millions of copies of DNA from a *very small amount of DNA* without amplifying DNA in bacteria. -requires primers (high GC content is optimal) complementary to DNA that flanks interest, nucleotides, DNA polymerase from Thermus aquaticus. needs heat to melt double helix. DNA of interest is denatured, replicated and cooled to reneal. repeated several times until enough copies are available for further testing

replication

*RNA primer*- DNA hook on to primer; (RNA can directly paired with parent strand) *primase* synthesizes short primer in 5' to 3' direction. plases ~ 10 nucleotide RNA primer to begin DNA replication. constantly being added to lagging strand becasue okazaki needs new primer *leading* strand requires only one (reality a few primers) *DNA polymerase III* (prokaryotes; adds nucleotides to growing daughter strand) or *DNA polymerases α, δ, ε* (eukaroytes; adds nucleotides to growing daughter strand) begin synthesizing daughter strand of DNA in 5' to 3'. incoming nucleotides are *5' dATP, dCTP, dGTP, and dTTP*. as new *phosphodiester bond* is made, *free pyrophosphate (PPi)* is released *DNA polymerase I* (prokaryotes; fills in gaps left behind after RNA primer excision) or *RNase H* (eukaryote; excises RNA primer) help remove RNA. *DNA polymerase I* (prokaryotes) or *DNA polymerase δ* (eukaroyes) adds DNA nucleotides where RNA primer had been *DNA ligase* seals end of DNA together creating continuous strand esp b/t okzaki fragments

watson-crick model

- 3D structure of DNA - deduced double helical nature and proposed specific base pairing as the copying mechanism - double helix- 2 linear polynucleotide chains of DNA are wound together in spiral orientation along common axis - key feature: two strands are antiparallel (opposite directions; one is 5' to 3' polarity down page and other is 5' to 3' polarity up the page) - key feature: sugar-phosphate backbone is on outside of helix with nitrogenous bases on inside -key feature: A to T (2 HB), G to C (3 HB- making them more stronger)

synthesis of daughter strands

-*DNA polymerases* - reads template and synthesizes daughter strandd; reads 3' to 5' while ONLY synthesizing complementary strand 5' to 3' from a 3' to 5' template. antiparallel orientation. -*leading strand* is copied in continuous fashion- same direction as replication fork *read 3'to 5'; complement synthesized 5' to 3'* -*lagging strand* copied in opposite direction; this side of fork, parental is 5' to 3' polarity. *okazaki fragments* are small strands that create space for DNA polymerase to fill in.

recombinant DNA

-DNA fragment from any source to be multiplied by either gene cloning or PCR. analyze and alter genes and proteins. provides reagents necessary for genetic testing- carrier detection and prenatal diagnosis of genetic diseases- useful for gene therapy

Transgenic and Knockout Mice

-DNA isolated and introduced into eukaryotic cells -transgenic mice- altered at germ line by introducing cloned gene into fertilized ova or into embryonic stem cells (clone gene: transgene; if it is disease-producing allele, mice can be used to study disease process from early embryonic development through adulthood) similar approach to knockout mice- gene intentionally deleted- study human diseases -different approaches to developing transgenic mice: -microinject the gene into nucleus of newly fertilized ovum; implant into surrogate mother and offspring will contain transgene in all cells including germ line cells; will be passed on to their offsprings. it coexists with own copies of genes not deleted. -useful for studying *dominant* gene effects b/c recessive cannot be controlled

origins of replication

-DNA unwinds here. generation of new DNA proceeds in both directions creating *replication forks* on both sides -bacterial chromosome- closed ds circular DNA with single origin of replication. 2 replication forks move away in opposite directions around circle then eventually meet producing 2 identical circular molecules of DNA -eukaryotic replication must copy more bases (slower process); each chromosome contains 1 linear molecule of dsDNA having *multiple origins of replication*- as it moves toward each other, *sister chromatids* are created and chromatids will remain connected at *centromere*

recombinant plasmid vector

-DNA vector contain at least one sequence recognized by RE. requires origin of replication and at lease 1 gene for antibiotic resistance to allow for selection of colonies with recombinant plasmids.

nucleosides

-a 5 carbon sugar (pentose) linked to a nitrogenous base - formed by covalently linking the base to C-1' of the sugar

Z-DNA

-another form of DNA -has zig-zag appearance - left handed helix -turns every 4.6nm and contains 12 base pairs -high GC o salt concentration may contribute to this formation - unstable

deoxyribonucleic acid (DNA)

-bulk of DNA found in chromosomes in nucleus of eukaryotic cells and some in mitochondria and chloroplast -macromolecule, polydeoxyribonucleotide composed of many monodeoxyribonucleoties linked together

denatured DNA

-disrupt HB and base-pairing resulting in melting of double helix into 2 single strands that have separated from each other -none of covalent links in backbone breaks commonly used to denature: heat, alkaline pH, chemicals (formaldehyde and urea) denatured ssDNA can be reannealed if denaturing condition is slowly removed annealing is important in PCR, detection of specific DNA sequences; require probe DNA is added to mixture of target DNA sequences when they bind to target DNA- may provide evidence of presence of gene of interest. = hybridization

mismatch repair G2

-encoded by genes *MSH2 and MLH1* which detect and remove errors introduced in replication that were missed during S phase of cell cycle. homologues of *MutS and MutL* in prokaryotes which serve a similar function

tumor suppressor genes (mutated ones are-like cutting breaks on car; no longer slow cell cycle))

-ex: p53 or Rb (retinoblastoma) inhibit cell cycle or participate in DNA repair processes -function to stop tumor progression and are called *antioncogenes* -result in loss of tumor suppression activity- promoting cancer. inactivation of both alleles is necessary for loss of function

aromatic

-examples of biological aromatic heterocycles -unusually stable ring system that adheres to specific rules 1. compound is cyclic 2. compound is planar 3. conjugated (alternating single and multiple bonds or lone pairs creating at LEAST ONE unhybridized p-orbital for each atom in ring) 4. compound has 4n +2 (n= integer) pi electrons. *Huckel's rule* ex: benzene -extra stability due to delocalized pi electrons which can travel throughout entire compound using available molecular orbitals making them fairly unreactive heterocycles are ring structures contain at least 2 different elements in rings. purines and pyrimidine contain N thus are imbued with exceptional stability. helps explain utility of nucleotides as molecules for storing genetic information

safety concerns

-increased resistance in viruses and bacteria can impact both humans and environment -is it ethical to test for life threatening disease and potentially terminate pregnancy based on results; impacts relatives and privacy concerns are raised -ethics of choosing individuals for specific traits

proofreading S

-part of *DNA polymerase* enzyme -the lack of stability in incorrect pairs is detected and excised and can be replaced with correct one. -looks at level of methylation- template strand has existed in cell longer than daughter so more heavily methylated. -very efficient and correcting most errors. DNA ligase does not have proofreading ability thus likelihood of mutations in lagging strand is higher

restriction enzymes (endonucleases)

-recognize ds DNA sequences; palindromic; isolated from bacteria -used to cleave DNA before electrophoresis and southern blotting and to introduce gene of interest into viral vector. -bacteria: act as restriction and modification system that protects bacteria from infection by DNA viruses; RE can be cut through backbones of double helix sticky ends from cuts due to restriction enzymes are advantageous in facilitating recombination of restriction fragments with vector DNA. vector of choice can be cut with same RE and allow fragment to be inserted directly into vector

purines and pyramidines (PURe As Gold= 2 rings)

-represent common bases in eukaryotes but there are exceptions seen in tRNA and some prokaryotes and viruses -Purines contains 2 rings in structure; adenine (A) and guanine (G); both found in DNA and RNA -Pyramidines contains only 1 rings; cytosine (C) and thymine (T) and uracil (U); cytosine in DNA and RNA, thymine only in DNA and uracil only in RNA

gel electrophoresis and southern blotting

-separate macromolecules by charge and size. DNA will migrate to anode because - charge. preferred gel is agarose gel- longer DNA strand, slower to migrate southern blot- detect presence and quantity of DNA strands in sample. cut by restriction enzymes and separated by gel electrophoresis. DNA fragments are carefully transferred to membrane. then probed with many copies of ssDNA sequences. probe will bind to complementary sequence and from dsDNA. probes labeled with radiosotopes or indicator proteins. -useful when searching for particular DNA sequence b/c separates fragments by length and probes for sequence of interest

nucleotide excision repair G1 and G2

-thymine dimers are eliminated from DNA by NER The process of removing and then correctly replacing a damaged segment of DNA using the undamaged strand as a guide. *excision endonuclease* makes nicks in phosphodiester backbone of damaged strand on both sides of thymine dimer and removes defective oligonucleotide -DNA polymerase fills in game 5' to 3' using undamaged strand as template. nick is sealed by ligase -correct lesions that are large enough to distort double helix

helicase

-unwinds DNA to generate 2 ss template strands ahead of *polymerase*. -*single stranded DNA binding proteins* will bind to unraveled strand to prevent both from reassociating of strands and degradation of DNA by *nucleases*. -as *helicase* unwinds- causes positive *supercoils* that strain strand- wrapping of DNA on itself as helical structure is pushed ever further toward telomeres during replication -*DNA topoisomerases*- negative supercoils to reduce risk of strand breakage. works ahead of helicase nicking strand to allow the realization of torsional pressure and resealing strands -during replication: parental strands serve as templates for generation of new daughter strands- *semiconservative* because 1 parental strand retained in each of 2 identical dsDNA

few differences in prokaryotic and eukaryotic DNA synthesis

1. 5 classic DNA polymerses in eukaroytic cells: α, β, γ, δ, ε -DNA polymerases α, δ, ε work to synthesize both leading and lagging strand, δ fills in gaps left behind when RNA primers are removed - γ replicates mitochondrial DNA - β and ε important to process of DNA repair - δ, ε assisted by PCNA protein which assembles into trimer to form *sliding clamp* which helps strengthen interaction b/t DNA polymerases and template strand

DNA sequences can be written in different ways

5'-ATG-3' 1. if written backwards, ends must be 3'-GTA-5' 2. position of phosphates may be shown: pApTpG 3. "d" may be used as shorthand for deoxyribose: dAdTdG -DNA: double stranded RNA: single stranded (exceptions are seen in viruses)

with exception of DNA polymerase's reading direction, everything in molecular bio is 5' to 3'.

DNA polymerase reads 3' to 5' but: DNA synthesis, DNA repair, RNA transcription, RNA translation occurs 5' to 3'

chromatin

DNA that makes up chromosome is wound around histones 5 histone (nucleoproteins) proteins, 2 copies of each: H2A, H2B, H3, H4 forms the core and about 200 BP of DNA are wrapped around to form nucleosome (prokaryotes lack these) H1 seals off DNA that enters and leaves nucleosome- adding stability to structure -most other nucleoproteins are acid soluble and stimulate processes like transcription

heterochromatin

Eukaryotic chromatin that remains highly compacted during interphase and is generally not transcribed. highly repetitive sequences

centromeres

Region of DNA found in the center of a chromosome. 1) Composed of heterochromatin, with high GC content. 2) Allows sister chromatin to remain connected there until microtubules pull them away in anaphase.

B-DNA

Right-handed helical structure of DNA that exists when water is abundant; the secondary structure described by Watson and Crick and probably the most common DNA structure in cells. -helix makes turn every 3.4nm and contains 10 bases within that span. major and minor grooves are seen between interlocking strands and are site of protein binding

gene therapy

The insertion of working copies of a gene into the cells of a person with a genetic disorder in an attempt to correct the disorder ex: SCID working copy of gamma chain into virus to transmit functional gene into human cells. gene delivery vectors are modified viruses since viruses naturally infect cells to insert their own genetic material. a portion of viral genome replaced with clone gene so virus can infect but not complete replication cycle. this poses risk of integrating near and activating host oncogene

chargaff's rule

[A]=[T] and [G]=[C], they pair up across from one another forming two strands also called base pairing. -total purines = total pyramidines

cancer cells

cells that grow and divide continuously at an unregulated pace; *metastasis*- migration to distant tissues by bloodstream or lymphatic system. accumulate mutations

euchromatin

contains genetically active DNA and appears light under light microscopy

base excision repair G1 and G2

cytosine deamination- loss of amino group results in conversion of cytosine to uracil DNA repair that first excises modified bases and then replaces the entire nucleotide -affected base recognized and removed by *glycosylase enzyme* leaving behind *apurinic/apyramidinic (AP) site*- abasic site which is recognized by *AP endonuclease* that removes damage sequence. DNA polymerase and ligase fill and seal strand -correct lesions small enough not to distort double helix

oncogenes (like gas pedal; promote cell cycle)

encode cell cycle-related proteins; mutated genes that cause cancer *proto-oncogenes*- before oncogenes are mutated src- named after sarcoma- category of CT cancers

hybridization

joining of complementary base pair sequences. DNA-DNA or DNA-RNA. uses 2 ss sequences and vital part of PCR and southern blotting

DNA libraries

large collections of known DNA sequences could equal to the genome of an organism DNA fragments are cloned through vectors and utilized for further study can be made of genomic DNA (include coding- exon and noncoding- introns) or cDNA (reverse transcribing mRNA; lacks introns and only includes genes expressed- expression libraries. -cDNA can be used reliably to sequence specific genes and identify disease causing mutations produce recombinant proteins or produce transgenic animals

backbone of DNA

made up of alternating sugar and phosphate, bases are attached to the deoxyribose or sugar -determines directionality of DNA and read 5' to 3' -nucleotides joined by 3'-5' phosphodiester bondes -phosphate group links 3' carbon of one sugar to 5' phosphate group of next incoming sugar in chain -phosphate carry negative charge, thus DNA and RNA have overall negative charge -5' end will have -OH or phosphate group bonded to C5 sugar -3' end will have free OH on C3 of sugar -read and written 5' to 3'

nucleotides

one or more phosphate groups are attached to C5 of nucleoside. -single strand of nucleic acids linked by phosphodiester bonds named according to # of phosphate present -adenosine di and triphosphate (ADP and ATP) gain name from # of phosphate groups attached to nucleoside adenosine high energy compounds b/c of repulsion between closel associated negative charges on phosphate grouls -building blocks of DNA

chimera

patches of cell that lack transgene (host blastocyst) and patches that have transgene

ribose

pentose sugar in RNA

telomere

repeating nucleotide (TTAGGG) at the ends of DNA molecules that do not form genes and help prevent the loss of genes some of sequences is lost in each round of replication and can be replaced by telomerase- (more highly expressed in rapidly dividing cells. shortening of telomeres- aging -high GC content creates exceptionally strong strand attractions at end of chromosomes to prevent unraveling

deoxyribose

ribose with 2'-OH group replaced by H then it is DNA

replisome (replication complex)

set of specialized proteins that assist DNA polymerases

DNA sequencing

the process of determining the precise order of nucleotides within a DNA molecule -template DNA, primers, DNA polymerase, 4 deoxyribonucleotides. and modified base dideoxyribonucleotide (lack 3'OH required for elongation) is added in lower concentrations- in which polymerase can no longer ad to chain. resulting in many fragments being terminated by modified base. separated by size using gel electrophoresis. last base can be read, in order b/c gel electrophoresis separates by size

DNA cloning

the production of multiple copies of a specific DNA segment -need a *recombinant vector* (bacterial or viral plasmids) that can be transferred to host after insertion of DNA of interest. grown in colonies and one with recombinant vector is isolated. ensure that recombinant vector includes gene for antibiotic resistance then grow this colony in large quantities. bacteria can be made to express gene of interest or lysed to reisolate replicated recombinant vector (process by restriction enzymes to release cloned DNA)

telomeres

to lengthen time that cells can replicate and synthesize DNA before necessary genes are damaged, chromosomes contain telomeres. -located at tip of chromosomes and consist of repetitive sequences with high GC content=telomeres can be slightly degraded between replication cycles without loss of function -protect chromosome from losing important genes from incomplete replication of 5' end of DNA strand