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GENETIC ANALYSIS OF THE CONSERVED DOMAINS OF MSC1, A CRITICAL DETERMINANT OF CHROMOSOME STABILITY IN FISSION YEAST

by
Cherise T. Bernard
B.S., Spelman College - 2005

Thesis Advisor: Nancy Walworth, Ph.D.
Graduate Program in Cellular & Molecular Pharmacology

RWJMS Research Tower, Room V-10
Piscataway

Thursday, April 26, 2012
10:00 a.m.


Abstract

The cell cycle insures that DNA replication, chromosome segregation and cell
duplication occur in an organized and efficient manner. Eukaryotic cells have evolved
checkpoint mechanisms to mediate these critical cell cycle events when cells are
exposed to conditions that damage DNA, impede DNA replication or compromise
attachment of chromosomes to the mitotic spindle. Inhibition of cell cycle checkpoint
mechanisms can result in cell death or genomic instability, a hallmark of cancer. A
genetic search for proteins that improve cell survival in fission yeast cells deficient
for the DNA damage checkpoint yielded Msc1 (multi-copy suppressor of Chk1). A
variety of studies suggest that Msc1 is important for chromosome stability. Mutants
harboring a deletion of the msc1 gene are viable, but exhibit genetic interactions with
components of the kinetochore complex such as Mis6 and Mis12, the microtubuleassociated
protein Dis1, and a mutant version of the centromere marker and histone
H3 variant CENP-A. Msc1 has also been shown to be a member of the Swr1 histoneremodeling
complex, an ATP-driven complex that incorporates the histone H2A
variant H2A.Z into nucleosomes. Consistent with this relationship, studies
demonstrate that Msc1 acts through H2A.Z to promote the survival of cells with a
compromised DNA damage checkpoint.
Msc1 contains a Jumonji N domain, a Jumonji C domain, and three PHD
fingers. These domains are often found in proteins with chromatin and histone
modifying capabilities, but the specific mechanisms by which the structural features
of Msc1 contribute to its role in genomic integrity are unclear. The PHD fingers of
Msc1 exhibit E3 ubiquitin ligase activity. The C-terminal PHD domains are also
important for association of Msc1 with the Swr1 complex. The goal of this research is
to gain further understanding of how Msc1 confers chromosome stability by exposing
additional critical functions of its conserved domains.
A systematic genetic analysis of Msc1 deletion mutants has uncovered the
need for the previously uncharacterized JmjN domain of Msc1 to preserve the
viability of cells containing a CENP-A mutant, cnp1-1. We also report that all three
PHD fingers are necessary to rescue cells with compromised kinetochore function.
Surprisingly, our data suggests that the presence of the JmjC domain may have an
inhibitory effect in this role. Finally, we demonstrate the importance of Msc1 PHD2
finger and its ubiquitin ligase activity in the genetic interaction with Dis1, a protein
that facilitates chromosome attachment to the mitotic spindle. Taken together, we
conclude that the domains of Msc1 contribute in a variety of ways to the maintenance
of genomic integrity and are collectively indispensable for its actions at the
centromere/kinetochore region.


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