Ch 13: DNA Replication Flashcards by Andrew Gierys

Meselson-Stahl Experiment

proved the semi-conservative model proposed by watson and crick

Bacteria are grown in Heavy N15, then are transfered to N14 media

when separeted in a density gradient, you should get a medium density band = hybrid band, and more and more light DNA (two bands one medium and one light band that gets thicker over time) after successive generations of growth in N14 media

conservative will only be heavy and light bands

dispervide is one medium band and progressinge gets lighter

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Semi-conservative replicaiton in Eukaryotes

Stainign with BrdU

Incubate in media with bromodeoxyuradine

these will stain differently than thymidine

after successinve replicaitons you will observe Harlequin chromosomes (half and half)

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Bacterial chromosome replicaiton

availability of mutants
=> temperature sensitive mutants identify critical proteins

in vitro DNA replicaitons
=> use purified cellualr componest

advantages
=> circular chromosomes with a single origin of repication

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Unwinidng problem

DNA gyrase (TopoII) removes positive supercoild ahead od DNA pol

because the unwinding process caused downstream supercoiliing

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Tempates and Nontemplates for Polymarease activity

double stranded or single stranded only will not work

need primers, overhangs, hairpins with overhangs, or nicks

purpose = need 3’ free end to prime the replicaiton

Reason = 3’ hydroxyl end performs a nucleophillic attack on the phosphodiester bond (alpha phosphate) of the nucleotriphosphate at the growing end

magnesium is needed as a cofactor to draw the hydrogen away from the oxyygen to increase its nucleophilicity

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lagging vs leading strand

because DNA is anti-parallel
3’ to 5’
5’ to 3’

lagging goes away from replicaiton fork, needs to polyermize in many fragments

leadig strand moves towards the replicaiton fork

DNA is always synthesized in the 5’ to 3’ direction (need that hydroxyl group to act as a nucleophile)

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Evidence for the presence of okazaki fragments

Sucrose density gradient experiment

longer the time, the longer the fragments

short time short fragments

short fragments disapear because they become ligated togetehr

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DNA binding proteins at the replicaiton fork

single stranded dna binding protiens to keep the replicaiton fork open

primase to make an RNa primer for DNA pol

DNA helicase to unwind

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DNA polymerases travel together

*** be able to explain the diagram????

DNA flipped into a loop, both enzymes are joined together so both strands of DNA travel in the same direction even though there is a laggin strand

polymerase releases lagging strand when okazaki fragment encountered

DNA pol rebinds laggin strand template farther along

knowns as the DNA pol III holoenzyme

figure 13-4

see clamp, clamp loader etc

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beta sliding clamp

polymerase held to DNA by clamp as it moves along the template

enzyme disengages from beta clamp cycles to a recently assembled clamp waiting at upstream region

docking site for dna pol

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model of sliding clamp complex

clamp leader threased onto dna helix

clamp loader binds with atp bound

once dna is bound, atp is hydrolyzed, clamp is released form loader and dna pol iii binds

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exonucleaase activites of DNA pol I

5’ to 3’ exonuclease activity
role in RNA primer removal

3’ to 5’ exonuclease activity
maintains accuracy, proof reading
=> can tell the geometry of the base pairing
=> TA and CG have a proper width and angles

matched = 11Ang, mismatch is aroung 10 Angstroms

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Activation of 3’ to 5’ exonuclease activity

incorrect nucleotides incorperated once for every 15^5 to 10^6 times

actual mutation rate is one in every 10^9 nucleotides

polymerase stalls when incorrect nucleotide incorperated
=> raying of end

in a different region of the enzyme that the polymerase activity

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DNA pol Klenow fragment

has the 5’ to 3’ pol and 3’ to 5’ exonuclease but not hte 5’ to 3’ exonuclease activty

dont want the 5’ to 3’ fragment in the lab sometimes

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Replicaiton in eukaryotic cells

multiple origins of replication
=> replicate DNA in small portions
====> replicons

timing of replication determinded by gene activyt and chromatin conformation
=> more active genes are replicated first
=> heterochromatin replicates last

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Yeast replicon

ARS = autominous replicating sequence (kinda like Ori)

ORC = origin of replicaiton complex

MCM complex = helicase enzymes

cdc6 and cdt1 = kinases, are required

cdk and ddk are also kinases

need a phosphorylation event to initate replicaiton

ORC remains assocated with ARS and MCM move outwards b/c they are helicases and unwind the dna

MCM proteins displaced from dna, exported to the nucleus (or degraded)

MCM are known as licensing factors and are neede for replication.

need to get rid of theses becuase only want to replicate DNA once per division

cell cycle phases

G1= gap 1
S= synthesis
G2= gap 2
M= mitosis

cell cycle regulation in yeast

cdc2 kinase is impornat to phosphorylate G1 cyclins
=> G1 to S

cdc2 to phosphorylated mitotic cyclins
=> G2 to M

MCM proteins and replicative stress

siRNA used to deplete Mcm proteins in yeast

depletion of Mcm leads to reduced cell proliferation

increaseed chromosome instability with depleated Mcm3

instability enhanced by treatment with aphidicolin (inhibits DNA replication)

5 euakryotic DNA polymareses

alpha associated with primase

beta DNA repair

gamma mt DNA

delta primary replication enzyme

epsilon repair

Eukaryotic Replication fork

PCNA = sliding clamp

RFC = clamp loader

RPA = SSB proteins

Nuclear Martix and DNA replication

DNA spooled through replicaiton complex

replication machinery is scaffolded to the nuclear matrix

localization of DNA replication

the replication machinery is stationaty in the nuclear matrix

replicaiton forks localized within 50 to 250 sites, called replicaiton foci

the clustering of replication forks may provide a mechanism for coordinating the replciaiton of adjacent replicons on individual chromosomes

replication sites distributed through the matrix

DAPI (blue) labels double stranded DNA

alpha BrdU (green) incorperated into newly synthesized DNA

PCNA (beta clamp) distribution changes in progression through S phase

early S-phase replicaiton associated with lamins a and c

DNA replication assocated with lamins

when lamins dissasemble during DNA replication (because the nuclar matix dissolves) they serve as binding sites for replication machinery

Lamins association

lamina associated domains (LADs) on chromosomes revealed using antibody technique

LAD-nuclear lamina (NL) contacts are established early in G1 phase

LADs on the distal 25 Mb of chromosomes contact NL first and then gradually detach

S-phase chromatin (replicating) shows transiently increasedlamin interactions

telomeres initally associated with the chromatin because as chromosomes are pulled to the metaphase plate they apperar to be closest to the nuclear lamina

Distribution of histone core complexes

(h3h4)2 tetramers reamin intact h2a/h2b dimers separate from each other figure 13-4b ???????????? histone proteins are epigenetically modified= methylation parental epigentic patterns need to be replicated

Epigenetic state fo repliated chromatic

DNa methylation patterns maintained in newly synthesized DNA histone modifications also maintains patterns on old histones guide modification of new histones examples => methylated h3k9 and acetylated h4k12 part of positive feedback loop modifications copied to histones of adjacent nucleosome

DNA repair pathways

nucleotide excision repair => transcription coupled pathway => global pathway Base excision repair mismatch repair double stranded break repair => non-homologous end joining => homologous end joining

Nucleotide escision repair

in response to pyrimidine [thymine] dimer covalent linkage of thymine dimers induced by exposure to uv irradiation "bulk adduct" lesion signaled by stalled RNA pol and CSB protein TFHIIH (XPB and XPD components ) unwind DNA (helicase) Transcription-coupled pathway XPC signals the global pathway unwinidng of DNA by XPB and XPD proteins and helicase subunits TFIIH nucleases cut on 5' (XPF-ERCC1) and 3' (XPG) sides filling of gap by DNA pol delta/ epsilon dan repair synthesis ligation

Xeroderma pigmentosum

deficient for repairign damage from uv light nucleotide excision repair deficiency related to cockayne syndrome deficient for repair of transcriptionally active dna

BAse excision repair

[deamination of cytosine can be induced by chemicals to make uracil] uracil-dna glycosylase recognizes uracil glycosylase cleaves base (glycosidic bond) AP endonuclease cleaves dna backbone phosphodiesterase activity of polymerase beta removes sugar phosphate from excised base polymerase beta fills gap by inserting cytosine linked with dna ligase

Detecting damaged dna in base excision repair

dna glycosylase (hOGG1) inspects base paired to cytosine (all GC pairs) base flipped out of dna duplex (orginized guanine) base cleaved form sugar guanine not recognized by active site (returned to dna)

mismatch repair

recognizes distortion in geometry of base paired nuclotides distinguishes between newly synthesized and parental strands new strand is not yet methylated parental strand is methylated = DNA total is hemi-methylated

Double strand breakage repair non-homologous end joining

1) non-homologous end joining => ku recognizes break, recruites DNA-PKcs and DNA liase IV is recruited to join together => some DNA is needed to be lost through exonuclease activity => not perfect time course of kI localization at sites of DSB formation (induced by laser microbeam) Ku localised at damged site but only for time of damage repair see video on nexus

watch videos on nexus

do it

Double strand breakage repair homologous end joining

genetic recombination (occurs in meiosis), and most often occures when homologues are paired up in meiosis. Can happen at other times tho use homogous chromosome as a template to repair and copy DNA double strand break in one chromosome widening of gap by exonuclease activity single strand invades homologous chromosome (rad51 protein) and displaces strand => D-loop 100% see video on nexus

replication and repair sometimes dna is not repaired

DNA sometiem snot repaired before it is replicated DNA pol stalls recruties specilized polymerase => polymerases small tract of nucleotides (translesion synthesis) example sPlymerase n (eta) => inserts two A nucleotides opposite thymine dimer

Cancer, DAN repair and DNA replicaiton

DNA mutaiton can result in malignancies colon cancer => 15% of cases linked to mutated genes for mismatch repair replication errors common in repeated sequences ex TGFbeta receptr

Instability in TGF-beta1 receptor gene

vascular disease is believed to be the result of escessive wound healing resoponse to chronic injuries => hypertension, smoking TGFbeta1 gene product controls regulatory sytesm that respond to cellular injury damage to recepotor is associated with decreased antiproliferative effect analysis of microstaellite instability in TFGbeta 1 receptor sgene in atherosclerotic tissue or cells cultured from plaque mutation causes frame shift in gene that leads to the development of the plaque