Semiconservative Replication DNA Replication Chapter 11 Wikipedia Kornberg’s DNA Polymerase I DNA Polymerase Reaction Requirements for DNA synthesis DNA Polymerase Mg++ dNTP single stranded DNA template Priming 3’ OH Consider how the active site descriminates among the dNTP’s. Other Enzymatic Activities Origin of Replication E. coli DNA Polymerases 9 mers- TTATTTCCAC Enzymatic Activities I II III . 5’-3’ synthesis + + + Pair 13mers GATCTCTTATTAG Required Primer + + + Order of Interaction 1. dnaA binds 9mers 3’-5’ Exonuclease + + + 2. dnaBC bind dnaA and 13mer -dnaB ATP dependent (Proofreading Activity) helicase 3. ssbp bind unwound DNA stabilizing it 5’-3’ Exonuclease 4. Gyrase (topoisomerase) relieves torsional stress (Replacement Activity) + - - . 5. Primase (dnaG) synthesizes short primers 6. DNA polymerase III initiates synthesis DNA Polymerase III mutation lethal – essential to replication DNA Polymerase I mutation viable – higher rate of mutation DNA Polymerase II mutation viable – function unknown 1 dnaA Discontinuous Replication DNA Polymerase synthesizes 5’-3’ only http://oregonstate.edu/instruction/bb492/figletters/FigH3.html Two Replication Forks Replication Fork http://oregonstate.edu/instruction/bb492/figletters/FigH2.html DNA Polymerase Clamp Replication Fork Improved Processivity http://www.wehi.edu.au/education/wehi-tv/dna/replication.html 2 Simultaneous Synthesis Eukaryotic Issues • Size of Genome • Ends of Linear Chromosomes Movie Size of Eukaryotic Genome Multiple Origins of Replication Genome Size – E coli 4.6 Mbp genome – Human 3330 Mbp genome (Haploid) Speed of Polymerase E. coli 100kb/min with two replication forks 23 min to copy genome Humans 1kb/min with 2 replication forks per chromosome http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAReplication.html 47.7 days to replicate genome typical S phase – 2-3 hours embryonic S phase 3 minutes Eukaryotes can vary origins during development Problem with linear chromosomes Telomerase http://www.steve.gb.com/images/science/telomerase.png 3 Telomerase Biology • Aging and Senescence • Telomerase and Cancer http://learn.genetics.utah.edu/features/telomeres/ 4.