Lecture22S10n - BIS101/Engebrecht Lecture22 Announcement I...

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BIS101/Engebrecht Lecture22 5/21/10 Announcement: I will hold office hours from 3:00-4:30 on the next two Wednesday in SLB3061. DSBR Model (cont) 1) The DNA sustains a double stranded break (either deliberately by an enzyme in the case of meiotic recombination or the result of a nick and replication, or external agents as discussed above). 2) The end of the break is processed by a 5’ 3’ DNA exonuclease to generate 3’-OH ending ss tails. This process is referred to as “resection” (meaning removal). 3) Homology recognition between DNA molecules occurs, this is mediated by specific pairing proteins: RecA of E. coli is the prototypical protein, Rad51 in eukaryotes. RecA forms a filament on the ssDNA and helps mediate Watson- Crick base pairing between either the sister chromatid or the homologous chromosome. 4) Once homology is found, ss tail invades the unbroken duplex and primes DNA synthesis from the 3’-OH. This forms heteroduplex DNA. One strand is broken and one strand is not broken. If you have introduced genetic markers and/or have DNA sequence differences between homologous chromosomes, can generate mispairs. 5) Using both ends in this homology search process will establish a physical junction between broken and unbroken molecules. Both 3-OH ends will prime DNA synthesis to copy the previously degraded DNA. Upon ligating the broken strands you generate an intermediate with 2 cross-stranded structures called Holliday junctions. 6) Branch migration moves these structures to the right and left. In E. coli this is mediated by the RuvAB proteins. These can be thought of as molecular motors. 7) The Holliday junction is resolved by a Holliday junction specific endonuclease (RuvC). If the two cleavage events occur in the same orientation there will not be exchange of genetic material of flanking markers (reciprocal exchange) however, if the two cleavage event occur in two
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Lecture22S10n - BIS101/Engebrecht Lecture22 Announcement I...

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