Rad51–Rad52 Mediated Maintenance of Centromeric Chromatin in <i>Candida albicans</i>

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-ACaCse4in 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.

绝大多数真核生物的着丝粒（centromere）位置确定并非仅依赖DNA序列。然而，目前尚未明确着丝粒身份的非遗传决定因素。尽管多种机制可单独或协同在表观遗传层面指定着丝粒，但该领域的多数研究均聚焦于一个通用因子——着丝粒特异性组蛋白H3变体CENP-A，其通常被视为着丝粒身份的表观遗传决定因子。尽管不同物种中CENP-A在着丝粒处的装载时机存在差异，但在多数酵母的着丝粒中，CENP-A的沉积与复制偶联发生在S期早期。着丝粒是致病性出芽酵母白色念珠菌（Candida albicans）中最早复制的染色体区域。本研究通过二维琼脂糖凝胶电泳（2-dimensional agarose gel electrophoresis）实验，在白色念珠菌中鉴定出了靠近早期复制型着丝粒CEN7的复制起点ORI7-LI与ORI7-RI。研究发现，复制叉会以依赖动粒（kinetochore）的方式在CEN7处停滞，且在缺失同源重组（homologous recombination, HR）蛋白Rad51与Rad52的情况下，复制叉停滞现象会减弱。删除ORI7-RI会显著减弱复制叉停滞信号，并提升变异7号染色体的丢失率。已有研究表明，同源重组蛋白Rad51与Rad52参与复制叉重启。共聚焦显微镜（confocal microscopy）观察显示，rad51与rad52突变体存在动粒簇解聚现象，这是动粒结构不完整的证据。通过染色质免疫沉淀（chromatin immunoprecipitation, ChIP）实验检测发现，在缺失Rad51/Rad52的情况下，着丝粒处的CENP-ACaCse4水平下降，进而导致动粒结构破坏。此外，蛋白质免疫印迹（western blot）分析显示，同源重组突变体中异位分布的CENP-A分子会发生降解，其降解模式与此前报道的其他动粒突变体一致。最后，免疫共沉淀（co-immunoprecipitation）实验证实，Rad51与Rad52可在体内与CENP-ACaCse4发生物理相互作用。综上，同源重组蛋白Rad51与Rad52可通过调控早期复制型着丝粒的程序性停滞位点处的CENP-ACaCse4水平，表观遗传维持着丝粒的功能。