Genetic control of the error-prone repair of a chromosomal double-strand break with 5’ overhangs in yeast

Abstract

ABSTRACTA targeted double-strand break introduced into the genome ofSaccharomyces cerevisiaeis repaired by the relatively error-prone nonhomologous-end joining (NHEJ) pathway when homologous recombination is not an option. A ZFN cleavage site was inserted out-of-frame into theLYS2locus of a haploid yeast strain to study the genetic control of NHEJ when the ends contain 5′ overhangs. Repair events that destroyed the cleavage site were identified either as Lys+colonies on selective medium or as surviving colonies on rich medium. Junction sequences in Lys+events solely reflected NHEJ and were influenced by the nuclease activity of Mre11 as well as by the presence/absence of the NHEJ-specific polymerase Pol4 and the translesion-synthesis DNA polymerases Pol σ and Pol 11. Although most NHEJ events were dependent on Pol4, a 29-bp deletion with endpoints in 3-bp repeats was an exception. The Pol4-independent deletion required TLS polymerases as well as the exonuclease activity of the replicative Pol DNA polymerase. Survivors were equally split between NHEJ events and 1 kb or 11 kb deletions that reflected microhomology-mediated end joining (MMEJ). MMEJ events required the processive resection activity of Exo1/Sgs1, but there unexpectedly was no dependence on the Rad1-Rad10 endonuclease for the removal of presumptive 3′ tails. Finally, NHEJ was more efficient in non-growing than in growing cells and was most efficient in G0 cells. These studies provide novel insight into the flexibility and complexity of error-prone DSB repair in yeast.