bsci110f13 l2&3 dnareplication

8/13/2019 BSCI110F13 L2&3 DNAreplication

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These results confirm the hypothesis of semi-conservative replication



Eukaryotic DNA Replicationis Semi-Conservative

In direct visualizationof BrdU labeling

After GeimsaStain /Fluro dyes



Bacterial DNA Replication

Starts at oriC sequence that acts as theORIgin of replication where many proteinsact to get things rolling BIDIRECTIONALLY

eg., in E. coli , dnaA encodes a replicationinitiator protein that recruits other proteinsto help separate the two strands.

Characterized through ts mutations

and in vitro studies

The active area where replicationOccurs is the REPLICATION FORK

In bacteria two RFs travel in oppositedirections around the circular chromos

(ORIs typically very A-T rich regions??)“Theta”

Structure



The UnWinding Problem (recall SC DNA!)

In Bacteria, DNA Gyrase (a topoisomerase) removes (+)

Supercoils Ahead of the RF



http://en.wikipedia.org/wiki/Image:DNA_replication.svg

MANY ENZYMES are involved in the DNA replication fork

As Helicase unwinds DNA at RF, the DNA ahead must rotate andbuild up TWISTS. Topoisomerases solve this by cutting DNA

backbones to remove knots and other entanglements

ssDNA tends to fold backand 2o structure-ssBPs

prevent this.



DNA Polymerase moves 3’->5’ ALONG THE TEMPLATEto construct a new strand that grows 5’->3’

One of the new strandsgrows toward

RF whilst another growsaway from the RF



The RF SupportsSemi-Discontinuous

Processes

The Leading strand is

synthesizedcontinuously

while

The Lagging Strand is

synthesized

Discontinuously

OkazkiFragments



The 4 BasicStepsof

Lagging Strand

DNAReplication



Many Proteins Are Involved in the DNA RF



http://en.wikipedia.org/wiki/Image:DNA_replication.svg

Lets start with DNA Polymerase



There are Multiple DNA Polymerases

All can catalyze 5’

-3’

directional DNA polymerizationas well as many other specific activities



The Nobel Prize in Physiology or Medicine for 1959 wasawarded jointly to Severo Ochoa and Arthur Kornberg

"for their discovery of the mechanisms in the biologicalsynthesis of ribonucleic acid and deoxyribonucleic acid”

DNA Polymerase I and it’s catalytic activity



Mechanisms of DNA Replication



The Replisome: a DNA Pol III Holoenzyme Complex

(non-catalytic/keeps DNA Association)

(the Catalytic units)

Core Pol II

anchors

Not part of replisomebut still v. important

Clamp proteins form a sliding clamp aroundDNA, helping the DNA polymerase maintaincontact with its template and therebyassisting with processivity. The inner face ofthe clamp enables DNA to be threadedthrough it. Once the polymerase reaches theend of the template or detects doublestranded DNA, the sliding clamp undergoes aconformational change which releases theDNA polymerase.

Clamp-loadingproteins are used to

initially load theclamp, recognizing the

junction betweentemplate and RNA

primers.

f l



An Overview of Bi-Directional

Semi-Conservative DNA Replication



Pol I 5’->3’ ExoNuclease is responsible for Removal of Lagging Strand RNAprimer while its 3’->5’ ExoNuclease acts in Repair of Mismatches.

“High Fidelity”



DNA Ligase Joins Okazki Fragments Togetherinto Complete DNA Strand



An Overview of Some of the Proteins Involved inProkaryotic and Eukaryotic DNA Replication



The Eukaryotic Replication Fork is More Complex(e.g. more Proteins)

But The Essential Functions are Unchanged

Pol is the primary DNA synthesizing

enz./Pol synthesizes the RNA-DNAprimers/PCNA (proliferating cell nuclearantigen) is the sliding clamp that isloaded onto the DNA by RFC (repfactorC)/RPA (rep.protein A) is SSB-likeprotein/FEN-1 (flap endo/exo nuclease)together with RNAse H1 degrades DNA-RNA primers.



Rfs Appear to Cluster at Discrete Non-random

Replication Foci

(RPA-HIS)



Termination and the Telomere Problem

When Replication Forks Meet the DNA Replication Machineryis Displaced from the Templates and Replication ENDs.

There is a problem though at the ends of linear chromosomes-a region known as the Telomere.

As the replication fork nears the end of theDNA, there is no longer enough template tocontinue forming Okazaki fragments. So the5' end of each newly-synthesized strandcannot be completed. Thus each of thedaughter chromosomes will have a shortenedtelomere.

It is estimated that human telomeres loseabout 100 base pairs from their telomericDNA at each mitosis, After 125 mitoticdivisions, the telomeres would becompletely gone- This is the Hayflick

Limit to somatic cell division-is this whysomatic cells typically die at this point?



Telomeres use The Telomerase RiboProtein Complexto Counteract Replication Associated Shortening



Chromatin Reassembly Is Very Rapid at RF(H3H4)2 tetramer remain intact andare distributed randomly along withnew (H3H4)2 units to daughter DNAstrands.

H2A/H2B dimers do not remaintogether and bind randomly to newand old (H3H4)2 tetramers



Next Lecture: DNA Repair

DNA Damage

Structure of the base-excision repair enzymeuracil-DNA glycosylase.

http://en wikipedia r /wiki/DNA repair

DNA Repair Enzymes

http://en.wikipedia.org/wiki/DNA_glycosylases




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