A model of the biochemical steps that lead to MMEJ, NHEJ, and SSA. Following resection, MMEJ, NHEJ, and SSA all anneal microhomologies located at either side of the break, followed by end processing including 3′ flap removal, gap filling, and ligation of broken ends to complete repair.

The role of Mre11 nuclease activity in MMEJ. (A) SLY19-mre11Δ was transformed with yeast centromeric plasmids expressing MRE11, mre11-H125N, mre11-3, mre11-P162S, or an empty vector. Survival frequency and the relative contribution of MMEJ and NHEJ to survival after induction of HO breaks were determined by analysis of junctional sequences observed in survivors. (B) Survival frequency of SLY19-exo1Δ, SLY19-exo1Δ mre11Δ, or SLY19-mre11Δ expressing additional Exo1 from a GAL promoter. An empty vector containing the GAL promoter was used as a control. Each value represents the average from at least three independent experiments ± standard deviation.

MMEJ requires multiple polymerases including two translesion bypass polymerases. Survival frequency (A) and the contribution of MMEJ and NHEJ to repair (B) among survivors after induction of HO breaks of strains lacking one or more nonessential DNA polymerases or KU70 were determined by analysis of junctional sequences observed in survivors. (C) Plasmids expressing POL4 or its mutant derivatives were introduced into SLY19-pol4Δ, and the frequency of MMEJ and NHEJ following induction of HO breaks was determined as in A. Each value represents the average from at least three independent experiments ± standard deviation.

MMEJ frequency in various mutants. (A) Cleavage at two HO sites in the MAT locus of SLY19 separated by ∼2 kb of URA3 sequence in opposite orientation generates noncomplementary breaks that are preferentially joined by Ku- and Rad52-independent MMEJ. The location of primers for PCR amplification and sequencing are shown by arrows. (B) Survival frequency after induction of HO breaks of each mutant strain (shaded bars) and the corresponding KU70 deletion derivatives (solid bars). The frequency of survival after an HO-induced DSB was calculated by dividing the number of colonies growing on YEP–GAL by the number of colonies growing on YEPD. Each value represents the average from at least three independent experiments ± standard deviation. (C) Junctional sequences observed in survivors of SLY19 and the mutant derivatives were used to calculate the frequency of repair events carried out by MMEJ or NHEJ. Typically, MMEJ involves >5 bp of imperfect microhomology, whereas NHEJ joins ends using <5 nt of complementary base pairing. The region spanning junctional sequences was PCR amplified with a set of primers [p2 and pX (A)] that anneal to the proximal and distal regions of the HO cleavage sites and align with the original sequence of SLY19. For each mutant, between 24 and 96 survivors were sequenced (see supplemental Figure 2).

Lack of Rad51 rescues the MMEJ deficiency of srs2Δ. Survival frequency following induction of HO breaks of srs2Δ, rad51Δ, and srs2Δ rad51δ strains (shaded bars) and the corresponding KU70 deletion derivatives (solid bars) were determined as described in Figure 2. The average from at least three independent experiments ± standard deviation is shown.

Roles of Sae2, Mec1, and Tel1 in MMEJ and NHEJ. Contribution of MMEJ and NHEJ to repair among survivors after induction of HO breaks of sae2Δ, tel1Δ, and mec1Δsml1Δ strains in A or sae2Δ and sae2Δ mre11Δ expressing additional Exo1 in B were determined as described in Figure 2. Survival frequency following induction of HO breaks in sae2Δ or tel1Δ derivatives of SLY1 in C and SLY18 in D, which carry one or two HO cleavage sites in direct repeat orientation in the MAT locus, were determined by plating cells on YEP–GAL normalized by the plating efficiency on YEPD. Each value represents the average from at least three independent experiments ± standard deviation. HO cleavage sites in the MAT locus in SLY1 or SLY18 are shown schematically above each graph. In E–H ChIP assays were used to assess the levels of RPA in sae2Δ or tel1Δ at the DSB using an anti-RPA antibody. Chromatin was isolated at the indicated time after galactose addition, crosslinked with formaldehyde, and fragmented by sonication. After immunoprecipitation and reverse crosslinking, purified DNA was analyzed by qPCR using three sets of primers that anneal 0.2 kb in F, 1 kb in G, and 5 kb in H to the DSB, as well as primers specific for the PRE1 gene situated on chromosome V as a control. PCR signals from each primer set at different durations of HO expression were quantified and plotted as a graph. IP represents the ratio of the RPA PCR signal before and after HO induction, normalized by the PCR signal of the PRE1 control. The positions of HO cut site and the location of primers are shown in E. Each point is the average of two separate experiments.