Summary Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes remains unknown. We monitored 5’-strand resection at inducible DSB ends and identified proteins required for two stages of resection: initiation and long-range 5’-strand resection. The Mre11-Rad50-Xrs2 complex (MRX) initiates 5’ degradation, whereas Sgs1 and Dna2 degrade 5’-strands exposing long 3’-strands at 4.4 kb/h rate. Deletion of SGS1 or DNA2 reduces resection and DSB repair by single strand annealing between distant repeats. Resection in the absence of SGS1 or DNA2 depends on Exo1. In exo1Δ sgs1Δ mutants the MRX complex and Sae2 in a stepwise manner generate only few hundred nucleotides of ssDNA at the break resulting in inefficient gene conversion and G2/M damage checkpoint arrest. We provide the first comprehensive model of the early steps of DSB repair in eukaryotes.

DNA double-strand breaks are normal consequences of cell division and differentiation and must be repaired faithfully to maintain genome stability. Two mechanistically distinct pathways are known to efficiently repair double-strand breaks: homologous recombination and Ku-dependent non-homologous end joining. Recently, a third, less characterized repair mechanism, named microhomology-mediated end joining (MMEJ), has received increasing attention. MMEJ repairs DNA breaks via the use of substantial microhomology and always results in deletions. Furthermore, it probably contributes to oncogenic chromosome rearrangements and genetic variation in humans. Here, we summarize the genetic attributes of MMEJ from several model systems and discuss the relationship between MMEJ and ‘alternative end joining’. We propose a mechanistic model for MMEJ and highlight important questions for future research.

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.

In Saccharomyces strains in which homologous recombination is delayed sufficiently to activate the DNA damage checkpoint, Rad53p checkpoint kinase activity appears 1 hr after DSB induction and disappears soon after completion of repair. Cells lacking Srs2p helicase fail to recover even though they apparently complete DNA repair; Rad53p kinase remains activated. srs2Delta cells also fail to adapt when DSB repair is prevented. The recovery defect of srs2Delta is suppressed in mec1Delta strains lacking the checkpoint or when DSB repair occurs before checkpoint activation. Permanent preanaphase arrest of srs2Delta cells is reversed by the addition of caffeine after cells have arrested. Thus, in addition to its roles in recombination, Srs2p appears to be needed to turn off the DNA damage checkpoint.

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