ICQ 6 Review

ICQ 6 Review - B acterial DNA Semiconservative model...

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Bacterial DNA Semiconservative model: culture in heavy then dark medium, remove at various times 2 bands: 1 heavy, 1 heavy and 1 light Bidirectional Θ structures Gyurasits and Wake- [H]thymidine to label replication bubble, heavily labeled segments at both ends Semidiscontinuos: both daughters grow 5’ 3’, growing into replication fork Experimental proof Okazaki fragments smaller in dut- mutant than dut+ parent, more uracil so more fragments when uracil is cut Ung- mutant, ½ of new DNA was fragments
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Quick stop mutant: stop DNA synthesis immediately after being switched from permissive to restrictive temperature Slow stop mutant: complete ongoing round of synthesis but stop after Origin replication & DNA unwinding Cis player: OriC, eukaryotes have many DnaA boxes:
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cannot start until ADP replaced by ATP DUE: unwinding protein, 3 tandem AT rich repeats Purpose of AT rich: unstable and breathing here so affects trans replication factors OriC high freq. of GATC: DAM methlaytes A after replication, only parental strand methylated SeqA: sequesters hemimethylated sites to prevent errant replication Helicase unwinding DUE: unwinds and allows DnaA to bind to top, DnaB to lower strand, initiation DnaA: binds ATP DnaG primase: RNA primers, act once for leading strand, repeatedly for lagging, opp direction of DnaB DNA polymerase III holoenzyme: recruited RNase H: removes unwanted RNA primer fragments IHF: stimulates DnaA binding, Fis inhibits it Processivity: enzymes that remain associated with template DNA polymerase III holoemzyme higher processivity than DNA polymerase I P III H needs ATP Domain structures Emergent property: domains have measurable activity (whole is greater than sum of the parts) When domains all together they may have another property unseen before Domain III of DnaA is similar to Cdc6, eukaryotic counterpart Different sequences may give the same structure Why do we care? Model for eukaryotic DNA, other AAA+ proteins, potential drug targets, other complex protein mechanisms
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Elongation Trans players DNA polymerase I: exonuclease activity, removes last ribonuc., adds nucleotides to connect okazaki fragments DNA polymerase III holoenzyme: makes leading strand 5’ 3’, and lagging 3’ 5’ DnaB helicase: unwinds DNA, 5’ 3’ DNA ligase: joins Okazaki fragments DNA gyrase/topoisomerase I: relieve torsional strain SSB: prevents nuclease attack lagging strand Sliding clamp: E. coli has β dimer, human PCNA trimer Increases holoenymes processivity Clamp loader: ATP energy to load sliding clamp to DNA dnaX: makes τ and makes γ (smaller protein) “ribosome screws up:” frame shift causes truncation Central dogma: Info from RNA not always meant to be copied precisely DNA polymerase III holo: 2 τ instead of γ
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This note was uploaded on 11/26/2010 for the course BIO 101 taught by Professor Rytasvilgalys during the Spring '09 term at Duke.

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ICQ 6 Review - B acterial DNA Semiconservative model...

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