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BIOB10Y Lecture 21; BIOB11 Lecture 3

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Biological Sciences
Aarti Ashok

Lecture #21 th Date: Tuesday, July 20 /2010 DNA Replication and Repair - DNA is a double helix with anti-parallel strands - Backbone is made of sugar-phosphate chains; in middle base-pairs - The structure of DNA suggests a ^copying_ mechanism; parent strands + new strands yield ^daughter DNAs_ Models proposed for DNA replication - Conservative model o daughter DNA composed of 2 new strands o parent strands join back together to form parent DNA - Semi-conservative model o Daughter DNA composed of 1 parent strand and 1 new strand - Dispersive model o Daughter DNA composed of pieces from both new strands and parent strands o Hybrid molecules (still relatively 50% parent and 50% new; even though they are patches from each) The Meselson-Stahl experiment - E.coli grown in medium with N Æcertain isotope of nitrogen (ammonium chloride = source of nitrogen) - Shifted to second medium - DNA extracted after 1 round of replication; centrifuged to separate by density o 20 mins - SNA extracted after 2 rounds of replication; centrifuged o 40 mins - Separated using a cesium chloride gradient; less dense at top, more dense at bottom - Found that semi-conservative model holds true for DNA replication DNA replication is semi-conservative - True for bacteria and eukaryotic cells A closer look at replication - Bidirectionality of DNA replication at each origin/bubble - In bacterial cells: o DNA polymerases replicate DNA; require a primer strand (=RNA strand needed to initiate replication) and all 4 deoxyribonucleotides to replicate DNA o DNA Pol III and DNA Pol I required; neither can synthesize DNA in the 3[ Æ 5[ direction; only synthesize in 5[ Æ 3[ direction The directionality of DNA replication at a replication fork - Leading strand synthesized very easily - Lagging strand synthesized in several steps; though not much time/rate difference o Synthesized in several small pieces each with own RNA primers o Each segment = Okazaki fragment Leading and lagging strand synthesis - DNA helicase unwinds DNA - Primase attaches RNA primer - Single-stranded DNA binding proteins (SSB) Æ ? Other proteins that aid in lagging strand synthesis - Over 10 proteins = part of ^replication complex_; function critical for process - DNA Pol I replaces the RNA primers with new DNA - DNA ligase closes gap between adjacent Okazaki fragments - Primase: primer synthesisÆDNA Pol III: elongation ÆDNA Pol I: primer removal + gap filling ÆDNA ligase: strand seal Leading and lagging strands are made by 2 DNA pol III molecules working together - Known as the ^true? Model_ of DNA replication A closer look at DNA pol III - Part of large complex of proteins known as ^replisome_ - 2 polymerases - 2 subunits that hold 2 polymerases together - 2 -Jclamps ( =sliding clamps) Æ keep polymerases associated with the DNA; donut-shaped
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