New Material DNA Replica on Cell Cycle Cancer DNA Replica on I Basic Problems Machinery DNA Replica0on in Mitosis mitosis important for growth development of mul cellular organisms accurate DNA rep must precede mitosis error rate 1 in 109 nucleo des per division genome of a cell copied once per cell cycle Problems Associated with Copying Duplex DNA 1 where to begin DNA synthesis ORI 2 unwinding of duplex DNA template 3 primer synthesis short ss sequence made 3 end 4 accurate copying of DNA 5 relief of superhelical tension in DNA occurs when DNA is opened up 6 resolu on of products need enzymes called topoisomerases topo I to ght tension that geTng DNA strands apart from each other aided by another topoisomerase topo II 7 end replica on of linear chromosomes ORI Ori Sequence required for initiation of DNA replication Ori can be thousands of bases long complicated Difficult to study mechanistically Virus model has been immensely useful Homo sapiens Globin gene cluster Figure 5 37 Molecular Biology of the Cell Garland Science 2008 Ori is recognized by Initiator Protein Tumor T antigen of SV40 virus Origin Recognition Complex ORC Replication Forks sequence required for ini a on of DNA rep can be thousands of bps long complicated di cult to study virus model most useful recognized by ini0ator protein tumor T an0gen of SV40 virus DNA virus uses host rep machinery ORC Origin Recogni on Complex Replica0on Fork Replication Forks region of transi on Replication fork is region of transition between the template DNA and newly synthesized nascent DNA b tw template DNA newly synthesized nascent DNA moves 5 3 direc on of DNA synthesis usually bi direc onal Figure 5 7 Molecular Biology of the Cell Replication forks move in 5 to 3 direction Direction of DNA synthesis Replication forks are usually bi Theta structure directional Figure 5 6 Molecular Biology of the Cell Garland Science 2008 Replicons Replicons Chromosomes are replicated in units termed replicons Properties of Replicons Replicons contain an origin 1 A chromosome has many replicons 2 Average human chromosome is about 150 million nucleotides long 3 Only a thousandth 0 1 of a chromosome is copied as part of a Replicons replicon chromosome replica on units proper es contain an origin a chromosome has many replicons average human chromosome is 150 million nucleo des long only 0 1 of a chromosome is copied as part of a replicon experiment Huberman Riggs incorporate 3H thymidine length spacing of replicons radioac ve T only labels DNA not RNA autoradiography pulse chase bi direc onal growth mul ple replicons Huberman and Riggs 1968 Incorporate 3H thymidine Autoradiography Length and spacing of replicons Pulse chase Bi directional growth DNA polymerase DNA Polymerase Reac0on nucleo0de polymeriza0on substrate must have free 3 OH DNA grows from 5 3 polarity DNA Polymerase the Reaction pyrophosphate released Figure 5 4 Molecular Biology of the Cell Garland Science 2008 deoxynucleoside monophosphate incorporated Nucleotide Polymerization Substrate must have free 3 OH DNA grows from 5 to 3 end Polarity Pyrophosphate is released deoxynucleoside monophosphate is incorporated Low error rate Mechanisms DNA Polymerase Uses Multiple Steps to Achieve High Accuracy for Replication Low Error Rate Mechanisms DNA Polymerase Uses Multiple Steps to Achieve High Accuracy for Replication Editing during Polymerization Replica0on Step 5 3 polymeriza on 3 5 exonucleoly c proofreading strand directed mismatch repair combined Errors per Nucleo0de Editing during 1 in 105 Polymerization 1 in 102 1 in 102 1 in 109 Figure 5 9 Molecular Biology of the Cell Garland Science 2008 edi ng during polymeriza on Exonuclease directed proofreading Figure 5 9 Molecular Biology of the Cell Garland Science 2008 Exonuclease directed proofreading ExonucleaseHDirected Proofreading Exonuclease in Polymerase Increases Accuracy of DNA Synthesis Wrong nucleotide added wrong nucleo de added 3 5 exonuclease 3 to 5 exonuclease 5 3 polymerase 5 to 3 polymerase Figure 5 8 part 2 of 2 Molecular Biology of the Cell Garland Science 2008 Deficiencies of DNA Polymerase De ciencies of DNA Polymerase 1 cannot recognize origins ORI 2 cannot unwind DNA to produce ss DNA make bubble 3 cannot ini ate synthesis of DNA need primer can extend pre exis ng RNA or DNA 4 torsional tension that builds up ahead of replica on fork is not removed by DNA polymerase cannot get around this alone other proteins help replicate chromosomes DNA Helicase template moves 5 3 on DNA Helicase Moves 5 to 3 direction on template Unwinds duplex DNA unwinds duplex DNA Hydrolyses ATP hydrolyzes ATP ss DNAs Single stranded DNAs Helicase hexamer at Replication Fork Figure 5 14 Molecular Biology of the Cell SSB SingleHStranded Binding Protein SSB SINGLE STRANDED BINDING PROTEIN SSB binds ss DNA prevents re hybridiza on of ss DNA keeps ss DNA straight prepares template for DNA primase interacts w DNA primase which makes primer EX RFHA Replica on Factor A Figure 5 16 Molecular Biology of the Cell Garland Science 2008 Binds single stranded ss DNA Prevents rehybridization of the single stranded DNA Prepares the template for DNA primase Interacts with DNA primase Example Replication Factor A RF A Primer Synthesis Primase DNA Polymerase Primase DNA Polymerase Primase interacts w SSB DNA helicase synthesizes primers doesn t need a primer to start ini ates DNA synthesis can incorporate ribonucleo des EX DNA polymerase Interacts with SSB and the helicase Synthesizes primers Initiates DNA synthesis Can incorporate ribonucleotides Example DNA Polymerase Figure 5 11 Molecular Biology of the Cell Garland Science 2008 Swivel SWIVEL topoisomerase SOLUTION relieves over winding in DNA ahead of replica on fork PROBLEM unwinding of DNA ahead of replica on fork produces torsional tension over winding SWIVEL The Problem Unwinding of DNA ahead of the replication fork produces torsional tension overwinding Swivel relieves overwinding in DNA ahead of the replication fork Swivel is a topoisomerase Figure 5 21 Molecular Biology of the Cell Garland Science 2008 Topoisomerase action Topoisomerase I Strand Passage Activity Topoisomerase I Topoisomerase I StrandHPassage Ac0vity Strand Passage Activity making breaking a bond so NO ENERGY needed Figure 5 22 part 2 of 2 Molecular Biology of the Cell Garland Science 2008 Figure 5 22 part 2 of 2 Molecular Biology of the Cell Garland Science 2008 SV40 Replication SV40 Replication topoisomerase II makes ds DNA breaks