Rad51-Rad52 mediated maintenance of centromeric chromatin in Candida albicans.
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.
Molecular Mycology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India.
Sreyoshi
S
Mitra
Departamento Ciencias Biomédicas Área de Microbiología, Universidad de Extremadura, Badajoz, Spain.
Jonathan
J
Gómez-Raja
Departamento Ciencias Biomédicas Área de Microbiología, Universidad de Extremadura, Badajoz, Spain.
Germán
G
Larriba
Department of Biotechnology, VBS Purvanchal University, Jaunpur, India.
Dharani Dhar
DD
Dubey
Molecular Mycology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India.
Kaustuv
K
Sanyal
D002176:Candida albicans / Q000235:genetics* / Q000378:metabolism; D002503:Centromere / Q000235:genetics* / Q000378:metabolism; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism; D047369:Chromatin Immunoprecipitation / Q000379:methods; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D059765:Homologous Recombination / Q000235:genetics; D051135:Rad51 Recombinase / Q000235:genetics* / Q000378:metabolism*; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics* / Q000378:metabolism*; D018741:Replication Origin / Q000235:genetics
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D005656:Fungal Proteins; D006657:Histones; D051721:Rad52 DNA Repair and Recombination Protein; D051135:Rad51 Recombinase