课件成果分子molecular biology course-3a.pptx

DNA replication;DNA Replication Models of DNA replication: Meselson-Stahl Experiment DNA synthesis and elongation DNA polymerases Origin and initiation of DNA replication Prokaryote/eukaryote models (circular/linear chromosomes) Telomere replication;DNA replication: accuracy!;Semiconservative;Alternative models of DNA replication:;1958: Matthew Meselson Frank Stahl’s Experiment Equilibrium density gradient centrifugation;1958: Matthew Meselson Frank Stahl’s Experiment Semiconservative model of DNA replication ;Results of the Meselson and Stahl experiment demonstrating semiconservative replication of DNA;Genetic evidence of semiconservative replication?;EMS, known to induce G:C → A:T transitions;1955: Arthur Kornberg Worked with E. coli. Discovered the mechanisms of DNA synthesis. Four components are required: dNTPs: dATP, dTTP, dGTP, dCTP (deoxyribonucleoside 5’-triphosphates) (sugar-base + 3 phosphates) 2. DNA template DNA polymerase (Kornberg enzyme) 4. Mg 2+ (optimizes DNA polymerase activity);Studying DNA Replication;DNA Polymerase I Discovery ;DNA synthesis can be assayed biochemically by the incorporation of a radioactive precursor, such as [a32P] dTTP, into acid-precipitable DNA as a function of time.;Studying DNA Replication;Pulse-Chase Assays ; Combining In Vitro Extracts With Genetics;Two Kinds of Conditional Mutants;In Vitro Complementation;Okazaki and colleagues obtained evidence for discontinuous DNA synthesis during replication in 1968. Molecules in the process of being synthesized can be labeled by introducing radioactively labeled precursor molecules for a brief period of time; this procedure is called a?pulse.;What would you?expect?to see if the replicating molecules were sedimented on a neutral (nonalkaline) sucrose gradient?;Pulse Chase Experiments reveal DNA is made in short pieces that e longer when combined.;Mutation Analysis Reveals Polymerase I Can’t Be the Only DNA Polymerase;Three main features of the DNA synthesis reaction: