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CHAPTER 12 – DNA REPLICATION AND RECOMBINATIONPreventing Train Wrecks in Replication- DNA polymerases are the enzymes that synthesize DNA- DNA replication – the process whereby a cell doubles its DNA before divisionThe Central Problem of Replication- Errors arise whenever information is copiedo The more times it is copied, the greater the number of errors- The copying of DNA must be accurate and must occur quicklySemiconservative Replication- The complementary nature of the two nucleotide strands in a DNA molecule suggest that during replication each strand can serve as a template for the synthesis of a new strand- Semi-conservative replication – replication in which each of the original nucleotide strands remains intact, despite no longer being combined in the same molecule- There were two alternative methods proposed for DNA replication:o Conservative replication – when the entire double-stranded DNA molecule serves as a template for a whole new molecule of DNAo Dispersive replication – when both nucleotide strands break down into fragments, which serve as templates for the synthesis of new DNA fragments, and then somehow reassemble into two complete DNA moleculesMeselson and Stahl’s Experiment- Used to determine which of the three models of replication applied to E. coli- Grew a culture of E. coli in a medium that contained 15N as the sole nitrogen source so that after many generations, all the E. coli cells would have 15N incorporated into the purine and pyrimidine bases of DNA- Took a sample of these bacteria and switched the rest to a medium containing only 14N and then took additional samples over the next few generations- Equilibrium density gradient centrifugation – when a centrifuge tube is filled with a heavy salt solution and a substance whose density is to be measured and spun for several dayso A density gradient forms, with high density at the bottom and low density at the top- DNA from bacteria grown only on medium containing 15N produced a single band at the position expected of DNA containing only 15N- DNA from bacteria transferred to the medium with 14N and allowed one round of replication also produced a single band but at a position intermediate between that expected of DNA containing only 15N and that expected of DNA containing only 14No Conservative model predicts one heavy band and one light bando These results were predicted by both semiconservative and dispersive- To distinguish between semiconservative and dispersive, bacteria were grown on medium containing 14N for a second generationo Two bands of equal intensity appeared, one in the intermediate position and the other at the position expected of DNA that contained only 14NModes of Replication- Replicons – individual units of replicationo Contain a replication origin- Replication begins at the origin and continues until the entire replicon has been replicatedo Bacterial chromosomes have a single origino Eukaryotic chromosomes contain many origins- Theta replicationo Theta replication – a common type of replication that takes place in circular DNA Double-stranded DNA begins to unwind at the origin, producing single-stranded nucleotide strands that then serve as templates on which new DNA can be synthesizedo Replication bubble – a loop formed from the unwinding of the double helixo Replication fork – the point of unwinding, where the two single nucleotide strands separate from the double-stranded DNA helixo Bidirectional replication – when there are two replication forks and they proceed outward in both directions, simultaneously unwinding and replicating the DNA until they eventually meeto Products are two circular DNA molecules- Rolling-circle replicationo Rolling-circle replication – a form of replication that takes place in some viruses and in the F factoro Initiated by a break in one of the nucleotide strands that creates a 3’-OH group and a 5’-phosphate groupo New nucleotides are added to the 3’ end of the broken strando Products are multiple circular DNA molecules- Linear eukaryotic replicationo Too much DNA to be replicated from a single origino Takes place simultaneously from multiple origins at a slower rateo At each replication origin, the DNA unwinds and produces a replication bubble Replication takes place on both strands at each end of the bubble, with the two replication forks spreading outward Eventually replication forks of adjacent replicons run into each other and the replicons fuse to form longs stretches of newly synthesized DNARequirements of Replication- Three major groups of components required for replication:o A template consisting of single-stranded DNAo Raw materials (substrates) to be assembled into a new nucleotide strand Deoxyribonucleoside triphosphates (dNTPs)- A deoxyribose sugar and a base attached to three phosphateso Enzymes and other proteins that read the template and assemble the substrates into a DNA molecule- A double-stranded DNA molecule must unwind to expose the bases- Nucleotides are added to the 3’-OH group of the growing nucleotide strandDirection of Replication- DNA polymerases – the enzymes that synthesize DNAo Can add nucleotides only to the 3’ end of the growing strand- DNA always elongates in the 5’-to-3’ direction- Continuous replication – the continuous synthesis of a new strand in the 5’->2’ directiono Leading strand – the strand exposed in the 3’->5’ direction that undergoes continuous replication- Discontinuous replication – the synthesis of a new strand in short, discontinuous burstso Lagging strand – the strand exposed in the 5’->3’ direction that is synthesized discontinuouslyo Okazaki fragments – the short lengths of DNA produced by discontinuous replication of the lagging strand Link together to form a continuous new DNA molecule- Rolling-circle replication only has continuous replication- Replication in the replication bubble of eukaryotic DNA is the same as in the theta modelThe Mechanism of Replication- Four stages:o Initiationo Unwindingo Elongationo TerminationBacterial DNA Replication- Many aspects are similar to eukaryotic replication but there are some important differences- Initiationo Single replication origino Initiator proteins – bind to the origin and cause a short section of DNAto unwind Allows helicase and other single-strand-binding proteins to attach to the polynucleotide strand- Unwindingo DNA

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Type: Lecture Note
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