Chromosomal replication origins inCandida albicansare genetically defined irrespective of their chromosomal context

Abstract

AbstractInitiation of DNA replication occurs at specialized regions along chromosomes called origins. The knowledge of replication origins is imperative to understand regulation of DNA replication. The properties of replication origins in the pathogenic budding yeastCandida albicansremain enigmatic in the absence of the knowledge of authentic chromosomal origins. Earlier, we identified centromere proximal (pCEN) chromosomal origins on chromosomes 5 and 7 ofC. albicans. Here, we identify another centromere-distal (dCEN) chromosomal origin by two-dimensional agarose gel electrophoresis corresponding to a previously reported autonomously replicating sequence (ARS), CARS2. We show that all the identified chromosomal origins are bound byC. albicanshomologs of conserved pre-replication complex proteins Orc2 and Mcm2. Previous reports from our lab and others have shown that there is a strong inter-relationship between centromere function and pericentric origin activity, suggesting that these origins are epigenetically regulated. However, we find that short intergenic regions corresponding to each of these origins functions as an ARS element in circular plasmids. Further, we use the novel strategy ofin vivogap repair to demonstrate that circular ARS plasmids can exist independentlyin vivoinC. albicans.Taken together, these results show that DNA sequence underlies the function of both centromere proximal and centromere distal chromosomal origins inC. albicans.