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Peter Houston, Eugenia Xu, James Broach. Dept. of
Molecular Biology, Princeton University, LTL 301, Princeton, NJ,
08540, United States of America.
Haploid wildtype yeast can change mating type as much as once every
generation. The HO endonuclease initiates the process by cutting at
MAT. This DNA break is then repaired by homologous recombination
with one of two donor sequences, HML or HMR. The different mating
types a and α are biased in the selection of donor: a chooses HMLα,
and α chooses HMRa. This nonrandom cell type dependent selection
ensures that cells switch their mating type instead of futile
replacement of the same mating type allele. A physical examination
of the pairing of MAT with HML and HMR should provide insight into
the process. Preliminary results show that large scale
preorganization of the chromosome does not occur. This result
suggests that sampling of HML and HMR may be occurring after the DNA
break and the choice of donor occurs at the commitment to
recombination. Perhaps the rate limiting step that controls
selection occurs during formation of a stable strand invasion
intermediate or at the recruitment of a competent replication
holoenzyme. The observation of pairing in single cells will allow us
to dissect these events through the gratuitous use of mutants and
drugs. Previous genetic analysis of donor preference has been
elusive since the assays of the process were too cumbersome. We have
developed a facile assay of donor selection that allows the global
genetic analysis of mutants that affect the process. Results of
these studies and their implications will be discussed.
Program Nr. 279C from 2004 Yeast meeting |
