Chapter 25 DNA Metabolism Recombination rearrangement of genetic information 25 1 DNA Replication Template a structure that would allow molecules to be lined up in a specific order and joined to create a macromolecule with a unique sequence and function DNA Replication Follows a Set of Fundamental Rules DNA Replication Is Semiconservative o Semiconservative replication template for the synthesis of a new strand producing two new DNA molecules each with one new strand and one old strand when each DNA strand serves as a o Meselson and Stahl experiment supported this using heavy nitrogen Conservative replication would not yield hybrid molecules Replication Begins at an Origin and Usually Proceeds Bidirectionally o Cairns experiment using radioactively labeled thymidine o Replication forks unwound and the separated strands are quickly replicated dynamic points where parent DNA is being o Both ends of the loop have active replication forks o Denaturation mapping colleagues that determined that replication always initiates at a unique point o Origin where replication loops initiate a technique developed by Ross Inman and DNA Synthesis Proceeds in a 5 3 Direction and Is Semidiscontinuous o 3 OH is where DNA is elongated o The strand serving as the template is read from its 3 end toward its 5 o Okazaki fragments o Leading strand short pieces of discontinuously replicated DNA end same direction as the replication fork movement replicated in the opposite direction of the replication fork movement the strand that is continuously replicated in the the discontinuously replicated strand that is o Lagging strand DNA Is Degraded by Nucleases Nucleases enzymes that degrade DNA o DNases nucleases specific for DNA Two broad classes of nucleases o Exonucleases o Endonucleases strand or molecule degrade nucleic acids from one end of the molecule can degrade at specific internal sites in a nucleic acid DNA Is Synthesized by DNA Polymerases DNA polymerase I a single polypeptide enzyme that can synthesize DNA The fundamental reaction of DNA synthesis is a phosphoryl group transfer 3 OH is the nucleophile Inorganic pyrophosphate is released Minimal change in free energy Two requirements for DNA polymerases o All DNA polymerases require a template o Polymerases require a primer a strand segment with a free 3 hydroxyl group to which a nucleotide can be added Primer terminus the free 3 end of the primer Process is faster when a polymerase adds more nucleotides without dissociating from the template Processivity dissociates the average number of nucleotides added before a polymerase Replication Is Very Accurate 3 5 exonuclease activity double checks each nucleotide after it is added o Removes the mispaired nucleotide removing of a mispaired nucleotide by the 3 5 exonuclease Proofreading activity E Coli Has at Least Five DNA Polymerases DNA polymerase II DNA polymerase III DNA polymerases IV and V are involved in an unusual form of DNA repair an enzyme involved in one type of DNA repair the principal replication enzyme or Klenow fragment the portion of DNA polymerase I left Large fragment when the 5 3 exonuclease domain is removed o Retains proofreading and polymerization activities o 5 3 exonuclease activity can replace a segment of DNA or RNA paired to the template strand in nick translation DNA Replication Requires Many Enzymes and Protein Factors DNA replicase system enzymes and proteins used in replication Helicases enzymes that move along the DNA and separate the strands replisome a complex of 20 or more different enzymes that relieve the topological stress caused by stabilize the separated strands synthesize short segments of RNA as primers Replication of the E coli Chromosome Proceeds in Stages o Use ATP Topoisomerases strand separation DNA binding proteins Primases DNA ligases seal nicks Three stages o Initiation o Elongation o Termination Initiation o R sites are five repeats of 9 bp that serve as binding sites for the key initiator protein DnaA o DNA unwinding element DUE o AAA ATPase a protein family to which DnaA belongs a region rich in A T base pairs Form oligomers and hydrolyze ATP relatively slowly o Dam methylase methylates the N6 position of adenine within GATC which triggers initiation Elongation o Parent DNA is first unwound by helicases and the topological stress is relieved by topoisomerases o Each separated strand is stabilized by SSB o Leading strand synthesis Primase synthesizes a primer at the origin Primer is synthesized in the direction opposite to that in which the DnaB helicase is moving DNA polymerase III complex adds nucleotides Continuous o Lagging strand synthesis Accomplished in Okazaki fragments RNA primer is synthesized by primase and DNA polymerase III binds to the primer and adds deoxyribonucleotides Primosome Primer is removed and replaced with DNA by DNA polymerase a complex of DnaB helicase and DnaG primase I o Termination The nick is sealed by DNA ligase Ter sequences signal termination Catenanes DNA circles linked topologically Replication in Eukaryotic Cells Is Both Similar and More Complex defined replication origins Replicators Pre replicative complexes initiation sites that form at the end of the M phase Licensing replication complex of the replicative helicase Minichromosome maintenance formation of the pre RC rendering the cell competent for pre RCs complexes found on replication MCM proteins form the heterohexameric ORC origin recognition complex a six protein complex that loads MCM2 7 helicase onto the DNA o Has five AAA ATPase domains among its subunits DNA polymerase properties in all eukaryotic cells a multisubunit enzyme with similar structure and o No proofreading 3 5 exonuclease activity o Believed to function only in the synthesis of short primers DNA polymerase extend primers o Has 3 5 proofreading exonuclease activity DNA polymerase replaces DNA polymerase in some situations like DNA synthesized at the ends of each chromosome to signal repair Telomeres termination Viral DNA Polymerases Provide Targets for Antiviral Therapy 25 2 DNA Repair Mutations Are Linked to Cancer Mutation Lesion unrepaired DNA damage permanent change in the nucleotide sequence of DNA o Substitution mutation the replacement of one base pair with another o Insertion or deletion mutations addition or deletion of one or more base pairs o Silent mutation negligible effect on the function of a gene if the mutation affects nonessential DNA or if it has a Ames test measures the
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