Genomic Integrity Determinants in Saccharomyces Cerevisiae
.
Dongli Huang (1), Douglas Koshland (1)
(1)
Department of Biology, Johns Hopkins University, Baltimore, MD 21218,
Department of Embryology, Carnegie Institution of Washington, Baltimore,
MD 21210
Double strand breaks (DSBs) in DNA lead to chromosome
deletions and rearrangements that are a hallmark of tumor cells.
Numerous studies have investigated how cells repair double strand
breaks. However, little is known about the proteins that prevent double
strand breaks from occurring in the first place. We call these types of
proteins as genomic integrity determinants (GID). To identify GID
mutants, we screened a collection of ~1900 temperature sensitive lethal
mutants for those that showed an elevated loss of telomere linked
markers on a 1.6Mb yeast artificial chromosome (YAC). 9 potential GID
mutants were identified. We have defined a DNA elongation factor
(DBP11), two DNA replication initiation proteins ORC3, ORC5 as GID
proteins. In the YAC assay, the rate of terminal deletions was increased
8-24-fold by mutations in DPB11, ORC3 and ORC5. Pulsed
Field Gel Electrophoresis analysis has shown that mutation in
DPB11 leads to DNA breaks at a specific region, which is
confirmed by chromosome I - IV translocation in dpb11cells. The
removal of origins from a YAC dramatically enhanced its rate of terminal
deletions in the dpb11mutant, suggesting that
dpb11generates DSBs through a persistent replication fork. In
contrast the removal of origins from the same YAC suppressed its rate of
terminal deletions in orc3and orc5 mutants, suggesting a
post-replication-initiation role of the origin recognition proteins in
genomic stability.
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