Saccharomyces Ku70, Mre11/Rad50, and RPA Proteins Regulate Adaptation to G2/M Arrest after DNA Damage

Abstract

Saccharomyces cells suffering a single unrepairable double-strand break (DSB) exhibit a long, but transient arrest at G2/M. hdf1 cells, lacking Ku70p, fail to escape from this RAD9/RAD17-dependent checkpoint. The effect of hdf1 results from its accelerated 5' to 3' degradation of the broken chromosome. Permanent arrest in hdf1 cells is suppressed by rad50 or mre11 deletions that retard this degradation. Wild-type HDF1 cells also become permanently arrested when they experience two unrepairable DSBs. Both DSB-induced arrest conditions are suppressed by a mutation in the single-strand binding protein, RPA. We suggest that escape from the DNA damage-induced G2/M checkpoint depends on the extent of ssDNA created at broken chromosome ends. RPA appears to play a key intermediate step in this adaptation.

Rif1 and Exo1 regulate the genomic instability following telomere losses

“…This hypothesis is difficult to test in PAL survivors directly, due to the on‐going erosion of chromosome ends. However, we tested this hypothesis at an HO‐induced double strand break (DSB) at the MAT locus in JKM139‐derived cells unable to repair the break by homologous recombination, as described previously (Lee et al ., 1998). Normally yeast Rif1 fails to significantly associate with a DSB (Xue et al ., 2011), whereas mammalian Rif1 co‐localizes with DSBs (Buonomo et al ., 2009).…”

“…(A) Association of Rif1‐ HA , expressed from the ADH 1 promoter, with the margins of a DSB in strains with a JKM 139 background, expressing the HO ‐nuclease from a galactose‐induced promoter (Lee et al ., 1998). Strains were grown overnight on raffinose; galactose was added at time 0 and samples collected every second hour.…”

“…Galactose induces GAL ‐ HO ‐nuclease to cut at the MAT a locus. Because the donor locus is missing, repair by recombination is prevented (Lee et al ., 1998). The numbers on the X ‐axis indicate the distance from the DSB .…”

“…Almost all cells arrested proliferation within 4 h following induction of the DSB, but later underwent checkpoint adaptation (e.g. they started to divide again), consistent with previous reports (Lee et al ., 1998; Pellicioli et al ., 2001). Up to 50% of the GAL‐HO cells (marked as wild‐type) adapted by 14 h after the DSB induction (Fig.…”

Breaking down cell cycle checkpoints and DNA repair during antigen receptor gene assembly

“…In yeast, the presence of a single irreparable DSB is not lethal: after initial checkpoint arrest, the broken chromosome can be replicated and persists for up to 10 cell divisions (Sandell and Zakian, 1993;Toczyski et al, 1997;Lee et al, 1998;Kaye et al, 2004). In checkpoint deficient strains (for example RAD9, RAD17 or MEC1 (similar to ATM/ATR)), cells do not even delay the first cycle and they continue to divide despite chromosome loss and genomic instability (Galgoczy and Toczyski, 2001).…”