MCB10_12 - Repair of a dsDNA break This process of strand...

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Unformatted text preview: Repair of a dsDNA break This process of strand invasion or strand exchange requires specialized factors that help the ssDNA region Fnd the homologous dsDNA. The broken chromosome 3 end(s) are used as primers for synthesis of DNA missing from the break. A second intact dsDNA provides the template. The process that creates stable strand exchange should require that only homologous regions are paired (i.e., two copies of the SAME GENE). 1 HR can be used to accurately repair a dsDNA break, if there is a similar copy of intact dsDNA to provide the correct sequence. Our cells are diploid, with two copies of each chromosome. Bacteria are haploid, one copy, but often have a second chromosome copy from ongoing DNA replication. STEP 1 create ssDNA with free 3OH STEP 2 Fnd homology by strand exchange: STEP 3 extend the region of strand exchange beyond the initial homology STEP 4 resolve the junction of dsDNAs to reestablish 2 separate chromosomes ssW1 dsC2-W2 ssW2 dsC2-W1 Homologous Recombination (HR) 2 STEP 1: Create ssDNA with free 3 OH 3 5 5 3 3 5 5 3 Eukaryotes load a 5-3 exonuclease at a dsDNA break (as shown in the previous slide). It would also be possible to begin with a nick on one strand ... and load a helicase at the nick to displace ssDNA. 3 Model for DNA strand exchange by E. coli RecA: (a) RecA with bound ATP has high afFnity for DNA. (b) DNA binding is cooperative, resulting in 5-3 polymerization of a Flament on the single-stranded DNA with 1 RecA per 3 nt. (c) A DNA duplex is taken up into the Flament and sampled for homology. (d) If the duplex is homologous, one of its strands is transferred to the single strand originally bound in the Flament. The other strand of the duplex is displaced. (e) ATP hydrolysis induces disassembly of the RecA Flament (not shown). STEP 2: Strand exchange to Fnd homology 4 The RecA flament Forces the dsDNA into a new helix geometry (18.6 bp DNA/helix turn) , which encourages the bases oF the dsDNA to ip between attempted pairings with two partner strands (part b below). Note that there is not ATP hydrolysis during strand exchange: ATP hydrolysis only mediates RecA assembly and disassembly From DNA. Base ipping allows the ssDNA to sample homology with the dsDNA. IF a long region oF exchanged pairing is created (at least 50 bp), one strand oF the original dsDNA can be displaced to leave the original ssDNA paired with the complementary strand oF the original dsDNA (part c below). 5 Human cells have a RecA-like protein for HR called Rad51. Rad51 needs the assistance of accessory proteins....
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MCB10_12 - Repair of a dsDNA break This process of strand...

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