Chapter 25 – DNA Metabolism- Recombination – rearrangement of genetic information25.1 DNA Replication- Template – a structure that would allow molecules to be lined up in a specificorder and joined to create a macromolecule with a unique sequence and functionDNA Replication Follows a Set of Fundamental Rules- DNA Replication Is Semiconservativeo Semiconservative replication – when each DNA strand serves as a template for the synthesis of a new strand, producing two new DNA molecules, each with one new strand and one old strando Meselson and Stahl experiment supported this using heavy nitrogen Conservative replication would not yield hybrid molecules- Replication Begins at an Origin and Usually Proceeds Bidirectionallyo Cairns’ experiment using radioactively labeled thymidineo Replication forks – dynamic points where parent DNA is being unwound and the separated strands are quickly replicatedo Both ends of the loop have active replication forkso Denaturation mapping – a technique developed by Ross Inman and colleagues that determined that replication always initiates at a unique pointo Origin – where replication loops initiate- DNA Synthesis Proceeds in a 5’->3’ Direction and Is Semidiscontinuouso 3’ OH is where DNA is elongatedo The strand serving as the template is read from its 3’ end toward its 5’endo Okazaki fragments – short pieces of discontinuously replicated DNA o Leading strand – the strand that is continuously replicated in the same direction as the replication fork movemento Lagging strand – the discontinuously replicated strand that is replicated in the opposite direction of the replication fork movementDNA Is Degraded by Nucleases- Nucleases – enzymes that degrade DNAo DNases – nucleases specific for DNA- Two broad classes of nucleases:o Exonucleases – degrade nucleic acids from one end of the moleculeo Endonucleases – can degrade at specific internal sites in a nucleic acid strand or moleculeDNA 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 templateo 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 – the average number of nucleotides added before a polymerase dissociatesReplication Is Very Accurate- 3’->5’ exonuclease activity double checks each nucleotide after it is addedo Removes the mispaired nucleotide- Proofreading – removing of a mispaired nucleotide by the 3’->5’ exonuclease activityE. Coli Has at Least Five DNA Polymerases- DNA polymerase II – an enzyme involved in one type of DNA repair- DNA polymerase III – the principal replication enzyme- DNA polymerases IV and V are involved in an unusual form of DNA repair- Large fragment or Klenow fragment – the portion of DNA polymerase I left when the 5’->3’ exonuclease domain is removedo Retains proofreading and polymerization activitieso 5’->3’ exonuclease activity can replace a segment of DNA (or RNA) paired to the template strand in nick translationDNA Replication Requires Many Enzymes and Protein Factors- DNA replicase system (replisome) – a complex of 20 or more different enzymes and proteins used in replication- Helicases – enzymes that move along the DNA and separate the strandso Use ATP- Topoisomerases – enzymes that relieve the topological stress caused by strand separation- DNA-binding proteins – stabilize the separated strands- Primases – synthesize short segments of RNA as primers- DNA ligases – seal nicksReplication of the E. coli Chromosome Proceeds in Stages- Three stages:o Initiationo Elongationo Termination- Initiationo R sites are five repeats of 9 bp that serve as binding sites for the key initiator protein DnaAo DNA unwinding element (DUE) – a region rich in A=T base pairso AAA+ ATPase – a protein family to which DnaA belongs Form oligomers and hydrolyze ATP relatively slowlyo Dam methylase methylates the N6 position of adenine within GATC, which triggers initiation- Elongationo Parent DNA is first unwound by helicases and the topological stress is relieved by topoisomeraseso Each separated strand is stabilized by SSBo Leading strand synthesis: Primase synthesizes a primer at the origin Primer is synthesized in the direction opposite to that in whichthe DnaB helicase is moving DNA polymerase III complex adds nucleotides Continuouso 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 – a complex of DnaB helicase and DnaG primase Primer is removed and replaced with DNA by DNA polymerase I The nick is sealed by DNA ligaseo Termination Ter sequences signal termination Catenanes – DNA circles linked topologicallyReplication in Eukaryotic Cells Is Both Similar and More Complex- Replicators – defined replication origins- Pre-replicative complexes (pre-RCs) – complexes found on replication initiation sites that form at the end of the M phase- Licensing – formation of the pre-RC, rendering the cell competent for replication- Minichromosome maintenance (MCM) proteins – form the heterohexameric complex of the replicative helicase- ORC (origin recognition complex) – a six-protein complex that loads MCM2-7 helicase onto the DNAo Has five AAA+ ATPase domains among its subunits- DNA polymerase - a multisubunit enzyme with similar structure and properties in all eukaryotic cellso No proofreading 3’->5’ exonuclease activityo Believed to function only in the synthesis of short primers- DNA polymerase - extend primerso Has 3’->5’ proofreading exonuclease activity- DNA polymerase - replaces DNA polymerase in some situations, like DNA repair- Telomeres – synthesized at the ends of each chromosome to signal terminationViral DNA Polymerases Provide Targets for Antiviral Therapy25.2 DNA
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