7 4 DNA Replication Precisely Duplicates the Genetic Material Replication is best studied in bacteria Though replication is very similar among Bacteria Archaea and Eukarya the processes are not identical The enzymes and proteins involved are parts of large complex aggregations of proteins and enzymes called replisomes These assemble at each replication fork Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 1 DNA Sequences at Replication Origins Replication origins have sequences that attract replication enzymes The origin of replication sequence of E coli is called oriC and it contains about 245 bp of A T rich DNA The origin is divided into three 13 bp sequences follwed by four 9 bp sequences Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 2 Bacterial Replication Origins Replication origins of bacterial species have similar conserved but not identical sequences Comparison between species leads to identification of consensus sequences the nucleotides found most often at each position of DNA in the conserved region The 13 mer and 9 mer sequences of oriC are conserved they play an essential role in replication Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 3 Eukaryote Replication Origins Saccharomyces cerevisiae yeast has the most fully characterized origin of replication sequences The multiple origins of replication are called autonomously replicating sequences ARS ARS organization and sequence is similar throughout the yeast genome characterized Replication origins of other eukaryotes are less well Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 4 Replication Initiation in Bacteria Replication in E coli requires that replication initiating enzymes locate and bind to oriC consensus sequences Enzymes DnaA DnaB DnaC bind at oriC and initiate DNA replication DnaA binds first bends the DNA and breaks hydrogen bonds in the A T rich sequences Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 5 Initiation of Replication DnaA first binds the 9 mer sequences bends the DNA and breaks hydrogen bonds in the A T rich sequences of the 13 mer region DnaB is a helicase that uses ATP energy to break hydrogen bonds of complementary bases to separate the strands and unwind the helix DnaB is carried to the DNA helix by DnaC Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 6 Initiation of Replication continued The unwound DNA strands are kept from re annealing by single stranded binding protein SSB Unwinding of circular chromosomes will create torsional stress potentially leading to supercoiled DNA Enzymes called topoisomerases catalyze controlled cleavage and rejoining of DNA that prevents overwinding Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 7 RNA Primers Are Needed for DNA Replication DNA polymerase elongates DNA strands by adding nucleotides to the 3 end of a pre existing strand They cannot initiate DNA strand synthesis on their own RNA primers are needed these are synthesized by a specialized RNA polymerase called primase Primase and some additional proteins join DnaA at oriC to form the primosome Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 8 Continuous and Discontinuous Strand Replication In E coli daughter DNA strands are synthesized by the DNA polymerase III pol III holoenzyme Holoenzyme refers to a multiprotein complex in which a core enzymes is associated with the additional components needed for full function The replisome is found at each replication fork and contains two copies of pol III Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 9 Leading and Lagging Strand Synthesis One copy of pol III synthesizes one daughter strand continuously in the same direction as fork progression this is the leading strand The other copy of pol III elongates the daughter strand discontinuously in the opposing direction to the fork progression via short fragments Okazaki fragments this is the lagging strand Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 10 RNA Primer Removal and Okazaki Fragment Ligation DNA polymerase I pol I uses two activities to complete replication Its 5 to 3 exonuclease activity removes the RNA primers Its 5 to 3 polymerase activity adds DNA nucleotides to the 3 end of the DNA segment preceding the primer DNA ligase seals the gap between the resulting DNA segments it does NOT add any bases though Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 11 Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 12 Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 13 Simultaneous Synthesis of Leading and Lagging Strands Each replisome complex carries out replication of the leading and lagging strand simultaneously The DNA pol III holoenzyme contains 11 protein subunits with the two pol III core polymerases each linked to a different copy of the tau protein The tau proteins are joined to a protein complex called the clamp loader two additional proteins form the sliding clamp Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 14 The Sliding Clamp The sliding clamp can close around the double stranded DNA during replication It has a doughnut hole 35 into which the DNA fits The sliding clamp anchors the DNA pol III core enzyme to the template It is required for the high level of pol III activity Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 15 Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 16 Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 17 DNA Proofreading Replication is very accurate DNA proofreading used to correct occasional errors Errors in replication occur 1 109 nucleotides in E coli Proofreading of DNA polymerase enzymes due to 3 to 5 exonuclease activity Several nucleotides including the incorrect one are removed and new nucleotides incorporated Genetics Analysis An Integrated Approach Copyright 2012 Pearson Education Inc 18 Eukaryotic DNA Polymerases Eukaryotes have many more DNA polymerases than bacteria DNA polymerase a alpha carries out synthesis of RNA primers Polymerase d delta and polymerase e epsilon carry out lagging and leading strand synthesis respectively Each interacts with