Rad51–Rad52 Mediated Maintenance of Centromeric Chromatin in Candida albicans

Abstract

Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-ACaCse4 levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-ACaCse4 in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENP-ACaCse4 levels at the programmed stall sites of early replicating centromeres.

Genetic interactions among homologous recombination mutants in Candida albicans

“…Recently, Rad51 and Rad52 have been implied in the deposition of the centromeric chromatin Cse4 and kinetochore assembly in C. albicans (Mitra et al, 2014). In support of this possibility, DAPI pictures from filamentous HR mutants very much resemble those of wild type (CAF2-1) cells treated with nocodazole, a drug that causes disassembly of mitotic spindles (Bai et al, 2002).…”

“…Alternatively, differences between both yeasts in DNA damage checkpoints (Harrison et al, 2014;Loll-Krippleber et al, 2014) or in the requirements for the activation of the spindle checkpoint could account for their differential ability to filament. S. cerevisiae carries DNA sequence dependent genetically determined point centromeres, whereas formation of C. albicans centromeres, and therefore in turn kinetochore-spindle microtubule interaction is sequence independent epigenetically regulated and centromeric chromatin deposition requires Rad51 and Rad52 (Mitra et al, 2014;Sanyal et al, 2004). It is unlikely that the increase in ploidy detected in filamentous HR mutants triggers filamentation, since fluconazoletreated C. albicans cells which also showed an increase in DNA content did not filament (Harrison et al, 2014).…”

Rad52 phosphorylation by Ipl1 and Mps1 contributes to Mps1 kinetochore localization and spindle assembly checkpoint regulation

“…In addition, some studies verified the involvement of components of the DNA damage pathway in the regulation of mitosis (35,56). Although the detailed mechanisms are not completely understood, it is evident that there exists a crosstalk between the DNA damage pathway and the regulation of mitosis.…”

A Surprising Role for the Sch9 Protein Kinase in Chromosome Segregation in Candida albicans

“…The strains were confirmed by Southern blot analysis ( Figure S1). We previously reported that the natural rate of loss of a chromosome in wildtype C. albicans (SN148) is ,5 3 10 24 /cell/generation (Mitra et al 2014). Two independent null (sch9/sch9) mutant strains (RMKS2A and RMKS2B) exhibited a 150-to 750-fold increase in chromosome loss as compared to the spontaneous rate of loss of a chromosome ( Figure 3B).…”

“…deletion of a centromere-proximal replication origin leads to a moderate increase in chromosome loss (Mitra et al 2014). In addition, phospho-regulation of kinetochore proteins by kinases (such as Aurora B kinase/polo-like kinase 1) has been shown to be critical for proper chromosome segregation (Shang et al 2003;McKinley and Cheeseman 2014).…”