Genome-wide studies of replication origins in Candida albicans reveal their conserved and distinct features

Abstract

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 function. 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.