2-3-11 Genome modification by HR

2-3-11 Genome modification by HR - February 3, 2011 Review...

Info iconThis preview shows pages 1–4. Sign up to view the full content.

View Full Document Right Arrow Icon
1 February 3, 2011 Modifying the genome by homologous recombination Review - Tuesday's Group Work: Suppose that homologous recombination is initiated at a double- strand break, and gives rise to the intermediate structure shown. Assume that no further branch migration takes place before the intermediate structure is resolved to yield complete recombination products. Which marker (if any) is likely to undergo gene conversion? If resolution comes about by cutting and rejoining of the strands indicated by arrows, will the products be crossover or non-crossover? Will the A allele be linked to the C allele, or to the c allele? A a B b c C Homologous recombination is found in all species - even those that don’t have sex (meiosis) - because it is needed to support replication . It is arguably the most complex of all DNA repair pathways, in terms of the different kinds of protein activities involved. The enzymology of homologous recombination is best understood in E. coli , but much is also known now about eukaryotes. Complex protein machines execute homologous recombination Processing of DNA ends by the E. coli RecBCD protein complex: The RecBCD complex enters from a broken end, and unwinds the strands, cutting the 3'ended strand frequently as it goes. When the complex comes to a Chi site (GCTGGTGG), the polarity of its endonuclease activity changes, so that the 5'- ended strand is cut more often, thus generating a 3'-ended ssDNA tail. The 3'-ended tail can then be bound by SSB or RecA .
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
2 RecA protein binds cooperatively to ssDNA RecA has remarkable abilities; it can: • Bind single-stranded DNA (ssDNA), to form a nucleoprotein filament , (This binding is "cooperative", i.e., self-propagating in the 5' to 3' direction.) • Seek out a homologous DNA sequence in another DNA duplex, • Pair the bound ssDNA strand with the complementary strand of the homologous duplex. See next figure… Experimental DNA substrates for assays of strand transfer by RecA in vitro This strand transfer,or "strand invasion" will occur only between homologous sequences. Three views of RecA - the E.coli strand transfer protein Top: electron micrograph of Rec A coating ssDNA molecules to form nucleoprotein filaments in vitro Middle: a space-filling model of the RecA/ssDNA nucleoprotein filament, showing its helical structure Bottom: an end-on view of six RecA molecules forming one helical turn of the filament (structure derived from X-ray crystallography) Homology testing by RecA involves two binding sites for DNA
Background image of page 2
3 Branch migration of Holliday junctions is catalysed by a protein "motor", the E.coli RuvA/RuvB complex RuvA is a structure-specific DNA-binding protein; it binds Holliday junctions. RuvB is an ATPase; it uses
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 4
This is the end of the preview. Sign up to access the rest of the document.

Page1 / 9

2-3-11 Genome modification by HR - February 3, 2011 Review...

This preview shows document pages 1 - 4. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online