Microhomology-mediated end joining in fission yeast is repressed by pku70 and relies on genes involved in homologous recombination

Abstract

Two DNA repair pathways are known to mediate DNA double-strand-break (DSB) repair: homologous recombination (HR) and nonhomologous end joining (NHEJ). In addition, a nonconservative backup pathway showing extensive nucleotide loss and relying on microhomologies at repair junctions was identified in NHEJ-deficient cells from a variety of organisms and found to be involved in chromosomal translocations. Here, an extrachromosomal assay was used to characterize this microhomology-mediated end-joining (MMEJ) mechanism in fission yeast. MMEJ was found to require at least five homologous nucleotides and its efficiency was decreased by the presence of nonhomologous nucleotides either within the overlapping sequences or at DSB ends. Exo1 exonuclease and Rad22, a Rad52 homolog, were required for repair, suggesting that MMEJ is related to the single-strand-annealing (SSA) pathway of HR. In addition, MMEJ-dependent repair of DSBs with discontinuous microhomologies was strictly dependent on Pol4, a PolX DNA polymerase. Although not strictly required, Msh2 and Pms1 mismatch repair proteins affected the pattern of MMEJ repair. Strikingly, Pku70 inhibited MMEJ and increased the minimal homology length required for efficient MMEJ. Overall, this study strongly suggests that MMEJ does not define a distinct DSB repair mechanism but reflects "micro-SSA."

Figure 1.—

Genetic requirements for fission yeast extrachromosomal MMEJ. (A) Presumed steps of MMEJ-dependent circularization of ura4⁺ gene. Two distinct repair junction sequences are predicted to arise following A/G mismatch correction: A:T and C:G. Position of the primers used to amplify repair junctions is shown (1: IPCRURA1small; 2: IPCRURA2). (B) MMEJ efficiency was calculated as the ratio of Ura⁺ colonies obtained after transformation with 2 μg (2+6) ura4⁺ substrate/Ura⁺ colonies scored after transformation with 1 μg REP4 plasmid (ura4⁺ PCR/REP4 molar ratio of 10). Four to 10 independent yeast transformations were performed in each mutant background. Error bars represent the standard error of the mean (SEM).

Figure 2.—

Mismatch correction at MMEJ repair junctions. (A) The frequency of mismatch correction to C:G was calculated for MMEJ repair junction sequences recovered from three independent yeast transformations with (2+6) ura4⁺ substrate. Error bars represent SEM. (B) MMEJ-associated mismatch correction was investigated in lig4Δ and lig4Δmsh2Δ cells following yeast transformation with ura4⁺ substrates presenting the following mismatches during repair: A/G [(2+6) ura4⁺], G/A, and A/A. Repair sequences were recovered from three independent yeast transformations (mean ± SEM). The total number of sequences analyzed is given under “n.”

Figure 3.—

DSB repair in nitrogen-starved cells. (A) Circularization efficiency of (2+6) ura4⁺ substrate through either NHEJ (WT-PN559) or MMEJ (lig4Δpku70Δ) was measured after three independent yeast transformations of either nitrogen-starved or exponentially growing cells. Relative circularization efficiency was calculated as the ratio of efficiencies in nitrogen-starved/exponentially growing cells. See legend of Figure 1 for the circularization efficiency measurement. Error bars represent SEM. (B) Examples of DSB repair junctions recovered from transformation of nitrogen-starved wild-type (PN559) cells with (2+6) ura4⁺ substrate. Microhomologous nucleotides are underlined. (C) A total of 63 DSB repair junctions recovered from exponentially growing wild-type (PN559) cells and 19 from nitrogen-starved wild-type cells were sequenced to determine nucleotide loss.

Figure 4.—

Repair of DSBs with continuous microhomologies. (A and B) ura4⁺ fragments with 3–8 terminal microhomologous base pairs were PCR amplified and used as EC MMEJ repair substrates in a series of lig4Δ derivatives. Circularization efficiency measurements with SEM (%) are detailed below the graph. (C) Circularization efficiencies of (2+6) and μ8 ura4⁺ repair substrates were measured in lig4Δ, lig4Δ pku70Δ, and exo1Δ cells. Three to 10 independent yeast transformations were performed in each mutant background and circularization efficiencies were calculated as described in the Figure 1 legend. Error bars represent SEM.

Figure 5.—

Nonhomologous nucleotides at DSB ends reduce MMEJ efficiency. (A) Four derivatives of μ8 substrate flanked by 1 [μ8(1)], 3 [μ8(3)], 5 [μ8(5)], or 7 [μ8(7)] nonhomologous base pairs were PCR amplified and used as EC MMEJ repair substrates in lig4Δ, lig4Δrad16Δ, lig4Δswi10Δ, and lig4Δpku70Δ cells. (B) Three derivatives of (2+6) repair substrate (I) containing 10 nonhomologous base pairs at the 5′-end (II), the 3′-end (III), or both ends (IV) were PCR amplified and introduced into lig4Δ and lig4Δpku70Δ cells. Three to 10 independent yeast transformations were performed in each mutant background and circularization efficiencies were calculated as described in the Figure 1 legend. Error bars represent SEM.

Figure 6.—

Relationships among MMEJ, HR, NHEJ, and mismatch repair pathways in fission yeast. Rad22 and Exo1 proteins are required for EC MMEJ, suggesting that MMEJ is related to SSA. Rhp51 is involved in EC MMEJ only if continuous microhomologies are available for the repair. Pol4 is strictly required for repair involving the use of discontinuous microhomologies. Msh2 MMR protein is also involved in EC MMEJ at discontinuous microhomologous regions. Both types of EC MMEJ (continuous/discontinuous microhomologies) are inhibited by Pku70.