DNA replication occurs only once in each mitotic cell
cycle. Tampering of the molecular mechanisms that impose this strict
regulation may lead to genetic instability, apoptosis, and cancer. Using
yeast as a model organism, we have taken several very different approaches
to address how this strict regulation at the initiation step is evolved
and the consequence of tampering with replication fork progression.
1. The evolution of replication origins by comparative functional
The replication machinery for the initiation of DNA synthesis is
well conserved from yeast to human, yet the sites at which the pre-replication
complex assembles appear to have diverged significantly between organisms
as well as between initiation sites within the same organism. Replication
origins of Saccharomyces cerevisiae are defined sequences of
about 100 bp that include a 17 bp essential element known as ARS consensus
sequence (ACS) where the origin recognition complex binds. Replication
origins of fission yeast are much larger and less well defined. In
mammals, replication initiation occurs in zones that require the support
of sequences as large as 50 kb. To better understand the
constituents of a replication origin, we have taken a comparative
functional genomics approach to analyze replication origins isolated
from different yeastspecies evolved pre or post genome duplication. This
approach allows us to address a number of probing questions such as:
- How are the ACSs of related yeastspecies different?
- How do the replication initiation machineries of the related yeast
species co-evolve with their respective ACSs?
- What are the additional essential elements for ARS function?
- Is there conservation in genomic locations for ARS function and
what is the evolutionary pressure?
- Does a phylogenetic tree derived from functional relatedness of
ARSs bear any resemblance to the phylogenetic tree derived from
genome sequence relatedness?
Investigator: Dr. Ivan Liachko
Collaborator: Dr. Uri Keich, Dept of Computer Science
2. Molecular mechanisms for the predisposition
to cancer by a defect in replication elongation
Genomic instability (GIN) and chromosome aneuploidy are hallmarks
of cancer cells, however their sources and functions in tumorigenesis
are unclear. DNA replication stress is a distinctive feature of uncontrolled
cell proliferation yet a causative relationship between DNA replication
defects and cancer has not been established. The F345I allele of Mcm4,
which encodes a subunit of the hexameric MCM helicase, induces a high
incidence (>80%) of mammary adenocarcinomas in homozygous female
mice. Homozygous diploid yeast carrying the equivalent point mutation
(mcm4Chaos3) are predisposed to genome instability and accelerated
proliferation as well as other characteristics of cancer cells including
gross chromosome rearrangements. Interestingly, haploid mutants
are not predisposed to these growth states or characteristics. We
will use the yeast model to address fundamental problems of tumor
progression and target specificity in cancer biology. Specifically,
we are investigating the mechanisms by which a defect in the replicative
helicase lead to accelerated proliferation, a universal characteristic
of cancer cells. We are also investigating the basis for the
distinctive responses of haploid and diploid cells to the mcm4Chaos3 mutation
that predisposes one of these cell types to cancer-like properties.
Investigator: Xin Li
Collaborator: Dr. John Schimenti
3. Organization and structure of the replication fork
unfaltering progression of every replication fork is vital to the
successful duplication of the genome. Mcm10 is a critical component
of the replication fork in coupling the MCM helicase with the elongation
machinery. Using Mcm10 as an entry point, we are studying the
structure and organization of the fork complex by genetic and biochemical
approaches. We have identified genetic interactors of Mcm10
by suppressor analysis and functional interactors of Mcm10 by conventional
biochemistry and in vitro reconstitution studies. Our goal
is to elucidate the process of assembly of the replication fork complex.
Investigators: Dr. Manhee Suh and Chanmi Lee
Collaborator: Dr. Shlomo Eisenberg of University of Connecticut,
Qiuyue Yang and Quan Hao of MacCHESS.