Centromeric R loops contribute to defects in kinetochore assembly and chromosomal instability

We mapped ssDNA formation in WT and hpr1delta cells at permissive (30C) and non-permissive (37C) temperature. We showed that hpr1 cells accumulate ssDNA show elevated levels of ssDNA at centromeric regions. Additionally, we show replication fork-associated ssDNA at origins of replication is more confined to the origins in hpr1 cells than WT cells, suggesting a defect in replication fork progression. We queried the yeast genome for ssDNA production in WT and hpr1delta cells. G1-arrested cells were released into S phase at 30C and 37C and collected at 10, 15, 20, and 25 min. Each S phase sample was paired with a G1 control sample and subject to ssDNA labeling with Cy3 and Cy5, respectively, prior to co-hybridization to the same microarray. The level of ssDNA was quantified as the ratio of Cy3 signals to Cy5 signals for every probe position on the microarray, after normalization.