Figure 1. Bre1 depletion leads to persistence of double-strand breaks (DSBs) and chromosomal instability (CIN). (A) Defect in homologous recombination due to depletion of Bre1 increases levels of DSBs, as shown by an increase in number of γH2AX-positive/Rad51-negative cells. CIN in Bre1-depleted cells manifests itself through a bridge-breakage-fusion cycle (B) and chromosomal aberrations, many of which involve heterochromatin (C). In (B) arrows point to anaphase bridges and arrowhead points to a micronucleus formed upon breakage of the bridge. In (C) the arrows point to chromosomes with centromeric heterochromatin amplification. (D) γH2AX co-localizes with heterochromatin of brightly-stained chromocenters (white arrows). Fixation for γH2AX, Rad51 staining, and chromosomal aberration analysis were performed on day 5 after Bre1 knockdown and were performed as described in ⁹. shBre1b, RNAi against Bre1b (Rnf40), shGFP, RNAi against GFP. (**p < 0.01, and ***p < 0.001, t-test).