CHAPTER 12 DNA REPLICATION AND RECOMBINATION Preventing Train Wrecks in Replication DNA polymerases are the enzymes that synthesize DNA DNA replication the process whereby a cell doubles its DNA before division The Central Problem of Replication Errors arise whenever information is copied o The more times it is copied the greater the number of errors The copying of DNA must be accurate and must occur quickly Semiconservative 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 nucleotide strands remains intact despite no longer being combined in the same molecule replication in which each of the original 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 DNA o 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 molecules Meselson 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 with a heavy salt solution and a substance whose density is to be measured and spun for several days when a centrifuge tube is filled o 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 14N o Conservative model predicts one heavy band and one light band o 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 generation o Two bands of equal intensity appeared one in the intermediate position and the other at the position expected of DNA that contained only 14N Modes of Replication Replicons individual units of replication o Contain a replication origin Replication begins at the origin and continues until the entire replicon has been replicated o Bacterial chromosomes have a single origin o Eukaryotic chromosomes contain many origins Theta replication o Theta replication circular DNA a common type of replication that takes place in 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 synthesized o Replication fork o Replication bubble a loop formed from the unwinding of the double helix the point of unwinding where the two single nucleotide strands separate from the double stranded DNA helix when there are two replication forks and they proceed outward in both directions simultaneously unwinding and replicating the DNA until they eventually meet o Bidirectional replication o Products are two circular DNA molecules Rolling circle replication o Rolling circle replication some viruses and in the F factor a form of replication that takes place in o Initiated by a break in one of the nucleotide strands that creates a 3 OH group and a 5 phosphate group o New nucleotides are added to the 3 end of the broken strand o Products are multiple circular DNA molecules Linear eukaryotic replication o Too much DNA to be replicated from a single origin o Takes place simultaneously from multiple origins at a slower rate o 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 DNA Requirements of Replication Three major groups of components required for replication o A template consisting of single stranded DNA o Raw materials substrates to be assembled into a new nucleotide strand Deoxyribonucleoside triphosphates dNTPs A deoxyribose sugar and a base attached to three phosphates o 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 strand Direction of Replication DNA polymerases the enzymes that synthesize DNA o Can add nucleotides only to the 3 end of the growing strand the continuous synthesis of a new strand in the 5 DNA always elongates in the 5 to 3 direction Continuous replication 2 direction undergoes continuous replication o Leading strand Discontinuous replication discontinuous bursts o Lagging strand the strand exposed in the 3 5 direction that the synthesis of a new strand in short the strand exposed in the 5 3 direction that is synthesized discontinuously discontinuous replication of the lagging strand o Okazaki fragments the short lengths of DNA produced by 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 model The Mechanism of Replication Four stages o Initiation o Unwinding o Elongation o Termination Bacterial DNA Replication Many aspects are similar to eukaryotic replication but there are some important differences Initiation o Single replication origin o Initiator proteins to unwind bind to the origin and cause a short section of DNA Allows helicase and other single strand binding proteins to attach to the polynucleotide strand Unwinding o DNA helicases enzymes that break the hydrogen bonds that exist between the bases of the two nucleotide strands of a DNA molecule Bind to the lagging strand template at each replication fork and move