When a centromere-containing plasmid containing two inverted
repeats of the E.coli LacZ gene, one of which has an HO endonuclease
recognition site, is induced to undergo double-strand break mediated
recombination, about 40% of the gene conversions have an accompanying
crossover. This is also the case with two yeast LEU2 sequence. However,
when two MAT-alpha sequences (one of which has a one-bp mutant to
prevent HO cleavage) are studied in the same plasmid context, only 8% of
the gene conversions are crossover-associated. A series of truncations
were performed to identify a region in the MAT sequences that impairs
crossing-over. When the cleavage site is surrounded by 30, 150 or 300 bp
on both sides, crossing-over rises to 30-50%, but crossing-over is again
lower when additional sequences are added on either side. Further
experiments are underway to identify the precise sequences that appear
to prevent crossing-over and to determine if this region will repress
crossing-over when introduced into the LacZ regions. From these
experiments we also show that the efficiency of homologous recombination
increases linearly from 60 to 600 bp on each side, and then levels off
as homology increases further. Surprisingly, similar dependence is found
when one side of the DSB has only 60 bp and the other has an increasing
amount of homology. This finding favors mechanisms that begin with
strand invasion on only one side of the DSB, as in several synthesis-
dependent strand annealing models.
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