Prashant K. Mishra, Arijita Chakraborty, Elaine Yeh, Wenyi Feng, Kerry S. Bloom and Munira A. Basrai

• Submitter: Prashant K. Mishra (Staff scientist, staff clinician)
• email: mishrap@mail.nih.gov

R-loops, the by-product of DNA-RNA hybridization and the displaced single stranded DNA, have been identified across the domains of life. The persistent presence of R-loops contributes to defects in DNA replication and repair, gene expression, and genomic integrity. R-loops have not been detected at centromeric (CEN) chromatin in wild-type budding yeast and its physiological significance in chromosome segregation remains unexplored. Here we used an R-loops accumulating hpr1∆ strain to investigate the consequences of R-loops at CEN chromatin and chromosome segregation. We show that Hpr1 interacts with CEN-specific histone H3 variant, Cse4 (CENP-A in humans), and prevents the accumulation of R-loops at CEN chromatin for chromosomal stability. DNA-RNA immunoprecipitation analysis showed an accumulation of R-loops at CEN chromatin in hpr1∆ strains, which was reduced by overexpression of RNH1. Increased levels of ssDNA, reduced levels of Cse4 and its assembly factor Scm3 (HJURP), and mis-localization of histone H3 at CEN chromatin were observed in hpr1∆ strains. Accumulation of R-loops at CEN chromatin contributes to defects in kinetochore biorientation and chromosomal instability (CIN), and these phenotypes were suppressed by RNH1 overexpression. In summary, our studies provide mechanistic insights into how accumulation of R-loops at CEN contributes to kinetochore dysfunction and CIN.