GDCB 511-4-24-09

GDCB 511-4-24-09 - GDCB 511: 24 April 2009 Homologous...

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GDCB 511: 24 April 2009 Homologous Recombination Weaver Chapter 22, 718 – 740 RecA RecBCD RuvA, B, C DSBR model for homologous recombination in eukaryotes Gene Conversion Yeast Mating Type Switching
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RecBCD: A substrate with a Chi site near one end was labeled (red dot) and incubated with RecBCD. The labeled DNA strand was nicked and the annealed oligo released, indicating that RecBCD has helicase activity. Experimental Support for the RecBCD Pathway
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A Synthetic Holliday Junction Mix oligonucleotides at annealing conditions for complementary base-pairing 5’-end of oligo 2 base-pairs with the 3’-end of oligo 1 5’-end of oligo 1 base-pairs with the 3’-end of oligo 2 Ends cross over in pairing
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Detecting a RuvA-RuvB-Holliday Junction Complex
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RuvA and RuvB form a DNA helicase that drives branch migration. RuvA tetramer (yellow) with square planar symmetry recognizes the center of a Holliday junction and binds to it. RuvB (blue) uses its ATPase to drive the DNA unwinding and rewinding necessary for branch migration.
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Resolution of the Holliday Junction Holliday junction can be resolved in 2 ways: Cuts 1 and 2 yield 2 duplex DNAs with patches of heteroduplex Cuts 3 and 4 yield crossover recombinant molecules with the 2 parts joined by a staggered splice
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Resolution: Holliday junctions are cleaved to release recombination products If “inner” strands are cut: gene conversion, no exchange of flanking markers. If “outer” strands are cut: reciprocal recombination, with exchange of flanking markers (cross-over).
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Resolution of Holliday junctions is catalyzed by the RuvC resolvase This protein acts as a dimer to clip 2 DNA strands to yield either patch or splice recombinant products Clipping occurs preferentially at the consensus sequence 5’-(A/T)TT (G/C)-3’ Branch migration is essential for efficient resolution of Holliday junctions Essential to reach preferred cutting sites RuvA, B, and C work together in a complex to locate and cut those sites
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Fig. 22.15 Low temperature, no MgCl2 37 degree and 5 mM MgCl2
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This note was uploaded on 03/08/2010 for the course GDCB 511 taught by Professor Yanhaiyin during the Spring '09 term at Iowa State.

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GDCB 511-4-24-09 - GDCB 511: 24 April 2009 Homologous...

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