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8/13/2019 BSCI110F13 L2&3 DNAreplication
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These results confirm the hypothesis of semi-conservative replication
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Eukaryotic DNA Replicationis Semi-Conservative
In direct visualizationof BrdU labeling
After GeimsaStain /Fluro dyes
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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
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The UnWinding Problem (recall SC DNA!)
In Bacteria, DNA Gyrase (a topoisomerase) removes (+)
Supercoils Ahead of the RF
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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.
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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
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The RF SupportsSemi-Discontinuous
Processes
The Leading strand is
synthesizedcontinuously
while
The Lagging Strand is
synthesized
Discontinuously
OkazkiFragments
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The 4 BasicStepsof
Lagging Strand
DNAReplication
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Many Proteins Are Involved in the DNA RF
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http://en.wikipedia.org/wiki/Image:DNA_replication.svg
Lets start with DNA Polymerase
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There are Multiple DNA Polymerases
All can catalyze 5’
-3’
directional DNA polymerizationas well as many other specific activities
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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
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Mechanisms of DNA Replication
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Mechanisms of DNA Replication
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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
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An Overview of Bi-Directional
Semi-Conservative DNA Replication
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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”
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DNA Ligase Joins Okazki Fragments Togetherinto Complete DNA Strand
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An Overview of Some of the Proteins Involved inProkaryotic and Eukaryotic DNA Replication
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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.
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Rfs Appear to Cluster at Discrete Non-random
Replication Foci
(RPA-HIS)
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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?
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Telomeres use The Telomerase RiboProtein Complexto Counteract Replication Associated Shortening
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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
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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