MCB 502A-2015. Lecture #7 !Chromosome replication: dna and pol mutants— Isolation of a polA mutant — Isolation and properties of DNA pol II and pol III— The “fork and knife” idea— DNA ligase— The Okazaki experiment— Semidiscontinuous DNA replication (uracil incorporation)? — RNA as a primer for DNA synthesisIsolation of a polA mutant-1— Last time …. — isolation of various dna(Ts)…. — The mutants that would completely stop … two major interesting types … at least ten different complementation groups (…). — … no change in … Kornberg polymerase. Immediate stop mutants identified functions responsible for replication fork progress. dnaB, dnaE, dnaF, dnaG, dnaL, dnaN, dnaQ and dnaX.Delayed-stop mutants identified functions responsible for replication initiation.dnaA and dnaC.Isolation of a polA mutant-2— … is Kornberg enz. the replicative polymerase?… … the same John Cairns … — … a replicative DNA polymerase should be an indispensable enzyme. — If cells can dispense with Kornberg DNA polymerase… — In vitro properties … — … John Cairns convinced Paula De Lucia … a mutant in Kornberg DNA polymerase. John CairnsIsolation of a polA mutant-3— … enrichments for dna-minus mutants … — … a brute force screen: … heavy mutageneis … … assay of individual clones … for activity of Kornberg DNA polymerase. — … simplification of the assay procedure, … to analyze 100 mutants a day ….By the way, do accelerate your research by simplifying your assays!Isolation of a polA mutant-4Sometimes a fishing expedition feels like a voyage into the unknown…— Paula … analyze 3,500 candidates … a single mutant… . #3478Isolation of a polA mutant-4— … the gene for the Kornberg DNA polymerase (was called) polA … and the isolated mutant received the designation polA1. — … a complete polA deletion would be inviable …. — … the polymerase activity inactivated, but the 5'—>3' exonuclease was intact… — … it grew almost normally … replication ….!5'—>3' pol3'—>5' exo5'—>3' exoIsolation of a polA mutant-5— …the mutant… established the non-replicative nature … — … an important lesson: to never assume that the actual in vivo function of the enzyme is what it does in assays in vitro! — As Genetics has to rely on Biochemistry …, Biochemistry has to rely on Genetics …— … Yin and Yang…. "Real polymerases"Kornberg polymeraseGeneticsBiochemistryIsolation and properties of DNA pol II and pol III-1— … a combination of polA mutant and Kornberg polymerase-specific antiserum … to look for novel DNA polymerase activities. — … two new DNA polymerases, … masked in the extracts of WT cells… — … Kornberg polymerase — DNA pol I, … DNA pol II and III. DNA pol IDNA pol II & IIIThe Kornberg dynasty— Curiously, purification and characterization of DNA pol II and III was done by Thomas Kornberg, the second son of Arthur Kornberg. Kornbergs - Roger, Ken, Sylvy, Arthur and Thomas in Stockholm, 1959Thomas Kornberg, UCSF, circa 1980Roger Kornberg, Stanford, circa 1970Kornbergs - Guy, Yahli Lorch, Roger, Gil and Maya in Stockholm, 2006Sigmund FreudOedipus complex!Isolation and properties of DNA pol II and pol III-2— … properties of the new polymerases … : — … optimal assay conditions of the new enzymes (salts, buffers, pH) …. direct assays … Pol I antiserum. The three DNA polymerases of E. coli: comparison.Functions pol I pol II pol IIIDNA synthesis 5’→3’ + + +DNA synthesis 3’→5’ - - -exonuclease 3’→5’ + + +exonuclease 5’→3’ + - -Template-primerIntact duplex - - -Primed single strands + - -Nicked duplex + - -Duplex with gaps <100 nt + + +Size, kD 109 88 140Molecules per cell 400 40 20DNA synthesis rate (nt/sec) 10 20 150Isolation and properties of DNA pol II and pol III-3— … gene for pol II — polB. — The double polA polB mutant …— … Pol II is slow … , … Pol III looked better… The three DNA polymerases of E. coli: comparison.Functions pol I pol II pol IIIDNA synthesis 5’→3’ + + +DNA synthesis 3’→5’ - - -exonuclease 3’→5’ + + +exonuclease 5’→3’ + - -Template-primerIntact duplex - - -Primed single strands + - -Nicked duplex + - -Duplex with gaps <100 nt + + +Size, kD 109 88 140Molecules per cell 400 40 20DNA synthesis rate (nt/sec) 10 20 150Isolation and properties of DNA pol II and pol III-4— … dna-(Ts) mutants … — … heat-liable DNA Pol III activity? — … the Pol III activity from dnaE mutants became inactive at 42°C. — Therefore, dnaE must code for Pol III… — … polC …..— Confusingly, … both dnaE and polC! The three DNA polymerases of E. coli: comparison.Functions pol I pol II pol IIIDNA synthesis 5’→3’ + + +DNA synthesis 3’→5’ - - -exonuclease 3’→5’ + + +exonuclease 5’→3’ + - -Template-primerIntact duplex - - -Primed single strands + - -Nicked duplex + - -Duplex with gaps <100 nt + + +Size, kD 109 88 140Molecules per cell 400 40 20DNA synthesis rate (nt/sec) 10 20 150B. subtilisIsolation and properties of DNA pol II and pol III-5— … the structure and function of DNA pol III later… … the two irritating universal properties of the E. coli DNA polymerases: 1) the inability to start DNA synthesis without a primer …; 2) the inability to synthesize DNA in the 3’—>5’ direction.The three DNA polymerases of E. coli: comparison.Functions pol I pol II pol IIIDNA synthesis 5’→3’ + + +DNA synthesis 3’→5’ - - -exonuclease 3’→5’ + + +exonuclease 5’→3’ + - -Template-primerIntact duplex - - -Primed single strands + - -Nicked duplex + - -Duplex with gaps <100 nt + + +Size, kD 109 88 140Molecules per cell 400 40 20DNA synthesis rate (nt/sec) 10 20 150Isolation and properties of DNA pol II and pol III-6— … instead of solving the mystery…, … , … … the two burning questions: 1) how is the DNA synthesis primed? 2) how is the 5’—>3’ DNA strand replicated?The “fork & knife” idea-1— … answeres to both questions … strange in vitro products … DNA pol I. — … instant (partial) renaturation … …branching …. — Kornberg’s interpretation… 5' 3'5' 3'5' 3'The “fork & knife” idea-2— … Walter Guild (1968) … elegant model …: Template switch Gap Repair Nick at Y-junctionSpecialized endonuclease— … "fork-and-knife" idea, …— …— … answers to both questions: 1) how
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