Initiation of DNA
replication in S. cerevisiae involves a homologous family of
proteins called the m ini c hromosome m aintainance
(Mcm) proteins. There are six of these proteins (Mcm2-7) and although
their function in initiation is not known, they have been shown to act
as a complex and interact with such proteins known to act at origins of
DNA replication as Orc and Cdc7/Dbf4. They are bound to chromatin in a
cell cycle dependent manner. Another Mcm protein, Mcm 10, was recently
characterized in our lab. Although identified in the same screen in
which some of the other Mcm proteins (Mcm2,3,5) were also identified,
Mcm10 is not a member of the homologous family. Its role in initiation
is also not well understood, but the protein binds single stranded DNA
and may be involved in several aspects of initiation and replication. I
will discuss an interesting genetic interaction between the mutants
mcm10-1 and cdc47-1 (an allele of
MCM7 ) which are both temperature sensitive. The double mutant is
viable at the restrictive temperature for both mutations, showing a kind
of mutual suppression . This mutual suppression is also seen
between mcm10-1 and a mutant allele of MCM 5 . I will
present a chracterization of the double mutant by 2D DNA gel, FACS
analysis, and MCM assay. I will also relate results of a two-hybrid
study and gene deletion to test possible hypotheses for the mechanism of
mutual suppression, and present a model to explain these fascinating
results. Understanding the cause of this mutual suppresion should shed
some light on the function of these Mcm proteins in DNA replication
initiation.
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