Saccharomyces cerevisiae Sae2- and Tel1-dependent single-strand DNA formation at DNA break promotes microhomology-mediated end joining - PubMed (original) (raw)

Saccharomyces cerevisiae Sae2- and Tel1-dependent single-strand DNA formation at DNA break promotes microhomology-mediated end joining

Kihoon Lee et al. Genetics. 2007 Aug.

Abstract

Microhomology-mediated end joining (MMEJ) joins DNA ends via short stretches [5-20 nucleotides (nt)] of direct repeat sequences, yielding deletions of intervening sequences. Non-homologous end joining (NHEJ) and single-strand annealing (SSA) are other error prone processes that anneal single-stranded DNA (ssDNA) via a few bases (<5 nt) or extensive direct repeat homologies (>20 nt). Although the genetic components involved in MMEJ are largely unknown, those in NHEJ and SSA are characterized in some detail. Here, we surveyed the role of NHEJ or SSA factors in joining of double-strand breaks (DSBs) with no complementary DNA ends that rely primarily on MMEJ repair. We found that MMEJ requires the nuclease activity of Mre11/Rad50/Xrs2, 3' flap removal by Rad1/Rad10, Nej1, and DNA synthesis by multiple polymerases including Pol4, Rad30, Rev3, and Pol32. The mismatch repair proteins, Rad52 group genes, and Rad27 are dispensable for MMEJ. Sae2 and Tel1 promote MMEJ but inhibit NHEJ, likely by regulating Mre11-dependent ssDNA accumulation at DNA break. Our data support the role of Sae2 and Tel1 in MMEJ and genome integrity.

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Figures

Figure 1.—

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.

Figure 2.—

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).

Figure 3.—

The role of Mre11 nuclease activity in MMEJ. (A) SLY19-_mre11_Δ 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-_exo1_Δ, SLY19-_exo1_Δ _mre11_Δ, or SLY19-_mre11_Δ 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.

Figure 4.—

Lack of Rad51 rescues the MMEJ deficiency of _srs2_Δ. Survival frequency following induction of HO breaks of _srs2_Δ, _rad51_Δ, and _srs2_Δ _rad51_δ 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.

Figure 5.—

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-_pol4_Δ, 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.

Figure 6.—

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 _sae2_Δ, _tel1_Δ, and _mec1_Δ_sml1_Δ strains in A or _sae2_Δ and _sae2_Δ _mre11_Δ expressing additional Exo1 in B were determined as described in Figure 2. Survival frequency following induction of HO breaks in _sae2_Δ or _tel1_Δ 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 _sae2_Δ or _tel1_Δ 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.

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Saccharomyces cerevisiae Sae2- and Tel1-dependent single-strand DNA formation at DNA break promotes microhomology-mediated end joining - PubMed (original) (raw)