Faithful DNA replication is required for genome inheritance during cell
division. To ensure complete duplication of the entire genome, the initiation of
replication occurs at multiple loci along the chromosomes. The determinants to
regulate where and when DNA replication initiates in higher eukaryotes are little
known. Not only the properties of nucleotide sequences, but also the dynamic
chromatin structure are highly controlled to define replication origins. The major
goal in my doctoral thesis is to understand the features of replication origins and
their impact on genome organization in the pathogenic yeast Candida albicans.
I combined computational genomics and experimental approaches to
address the following research objectives. First, I investigated the distinct
replication features of centromeres. Centromere is a specialized chromosomal
locus required for chromosome segregation during cell division. Importantly,
Candida centromeres are epigenetically defined regional centromeres, similar to
centromeres in higher eukaryotes. I found that centromeres constitutively
replicate first on each chromosome and this early replication event is linked to
the epigenetic nature of centromeres. Furthermore, aligning ORC binding sites
with conserved nucleosome depletion patterns throughout the genome revealed
the locations of potential chromosomal origins. Strikingly, origin DNA conferred
ARS (autonomously replication sequences) function on a linear plasmid vector.
Thus, I performed a genome-wide ARS screen to identify the consensus
sequences (ACS) for ARS function, and I identified a unique 15 bp ACS motif.
This motif is required for origin activity on the plasmid, and it functions in the chromosomal context when associated with appropriately positioned
nucleosomes. Thus, despite the presence of a regional, sequence-independent
centromere, C. albicans requires a specific sequence motif for replication origin
Taken together, I comprehensively mapped the origins of replication in the
C. albicans genome and characterized their conserved and distinct features.
Importantly, the discovery of the ACS motif allowed us to develop the first
plasmid shuttle vector for use as a genetic tool in C. albicans. All work here
facilitates the study of C. albicans as a promising model organism for
understanding fungal pathogenesis and eukaryotic genome organization.
University of Minnesota Ph.D. dissertation. April 2013. Major: Molecular, cellular, developmental biology and genetics. Advisors: Judith Berman, PhD, Duncan Clarke, PhD. 1 computer file (PDF); vii, 203 pages.
Genome-wide studies of replication origins in Candida albicans reveal their conserved and distinct features.
Retrieved from the University of Minnesota Digital Conservancy,
Content distributed via the University of Minnesota's Digital Conservancy may be subject to additional license and use restrictions applied by the depositor.