R-loops at centromeric chromatin contribute to defects in kinetochore integrity and chromosomal instability in budding yeast

Abstract

R-loops, the byproduct of DNA-RNA hybridization and the displaced single-stranded DNA (ssDNA), have been identified in bacteria, yeasts, and other eukaryotic organisms. 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 (<i>CEN)</i> chromatin in wild-type budding yeast. Here we used an <i>hpr1∆ </i>strain that accumulates R-loops to investigate the consequences of R-loops at <i>CEN</i> chromatin and chromosome segregation. We show that Hpr1 interacts with the <i>CEN</i>-histone H3 variant, Cse4, and prevents the accumulation of R-loops at <i>CEN</i> chromatin for chromosomal stability. DNA-RNA immunoprecipitation (DRIP) analysis showed an accumulation of R-loops at <i>CEN</i> chromatin that was reduced by overexpression of <i>RNH1</i> in <i>hpr1∆</i> strains. Increased levels of ssDNA, reduced levels of Cse4 and its assembly factor Scm3, and mislocalization of histone H3 at <i>CEN</i> chromatin were observed in <i>hpr1∆</i> strains. We determined that accumulation of R-loops at <i>CEN</i> chromatin contributes to defects in kinetochore biorientation and chromosomal instability (CIN) and these phenotypes are suppressed by <i>RNH1</i> overexpression in <i>hpr1∆</i> strains. In summary, our studies provide mechanistic insights into how accumulation of R-loops at <i>CEN</i> contributes to defects in kinetochore integrity and CIN.