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

R-loops 是 DNA-RNA 杂交的副产物和置换的单链 DNA，已在细菌、酵母和其他真核生物中被鉴定出来。R 环的持续存在导致 DNA 复制和修复、基因表达和基因组完整性的缺陷。在野生型出芽酵母的着丝粒 ( CEN)染色质中未检测到 R 环。在这里，我们使用了一个累积 R 环的hpr1Δ 菌株来研究 R 环在CEN染色质和染色体分离中的后果。我们表明 Hpr1 与CEN组蛋白 H3 变体Cse4 相互作用，并防止 R 环在CEN处积累染色质的染色体稳定性。DNA-RNA 免疫沉淀 (DRIP) 分析显示 R 环在CEN染色质上的积累，通过在hpr1Δ菌株中过表达RNH1而减少。在hpr1Δ菌株中观察到ssDNA 水平增加、Cse4 及其组装因子 Scm3 水平降低以及组蛋白 H3 在CEN染色质上的错误定位。我们确定在R-循环的积累CEN染色质有助于动粒biorientation和染色体的不稳定性（CIN）的缺陷，并且这些表型都受到抑制RNH1过度表达hpr1Δ菌株。总之，我们的研究提供了关于CEN中 R 环的积累如何导致动粒完整性和 CIN 缺陷的机制见解。