Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells

Abstract

Microhomology-mediated end joining (MMEJ) is a major pathway for Ku-independent alternative nonhomologous end joining, which contributes to chromosomal translocations and telomere fusions, but the underlying mechanism of MMEJ in mammalian cells is not well understood. In this study, we demonstrated that, distinct from Ku-dependent classical nonhomologous end joining, MMEJ--even with very limited end resection--requires cyclin-dependent kinase activities and increases significantly when cells enter S phase. We also showed that MMEJ shares the initial end resection step with homologous recombination (HR) by requiring meiotic recombination 11 homolog A (Mre11) nuclease activity, which is needed for subsequent recruitment of Bloom syndrome protein (BLM) and exonuclease 1 (Exo1) to DNA double-strand breaks (DSBs) to promote extended end resection and HR. MMEJ does not require S139-phosphorylated histone H2AX (γ-H2AX), suggesting that initial end resection likely occurs at DSB ends. Using a MMEJ and HR competition repair substrate, we demonstrated that MMEJ with short end resection is used in mammalian cells at the level of 10-20% of HR when both HR and nonhomologous end joining are available. Furthermore, MMEJ is used to repair DSBs generated at collapsed replication forks. These studies suggest that MMEJ not only is a backup repair pathway in mammalian cells, but also has important physiological roles in repairing DSBs to maintain cell viability, especially under genomic stress.

Keywords: BLM/Exo1; CtIP; DNA damage; DNA repair pathway; genome stability.

Fig. 1.

The nuclease activity of Mre11 is important for MMEJ. (A) EGFP-MMEJ DSB repair substrate. (B–E) U2OS cells with single integration of EGFP-MMEJ or EGFP-HR substrate and stably expressing shRNAs against Ku70 and/or Mre11 or No, with or without stably expressed Myc- or HA-tagged Mre11 (WT or H129N), as indicated (Fig. S4), were induced by I-SceI and assayed. For this figure and all of the following figures in the main text, data shown are mean of three independent experiments, with error bars as SD and P values as noted: *P ≤ 0.05; **P ≤ 0.01; and ***P ≤ 0.001.

Fig. 2.

MMEJ increases when cells enter S/G2. (A) Inducible I-SceI construct, DD-HA-I-SceI-GR. Stabilization (immunoblot) and localization (immunostaining) after Shield1 (2 h) or TA (15 min). (B) T98G cells with single integration of MMEJ substrate were asynchronized, G0/G1-arrested, or G0/G1-arrested and released and assayed. (C) Experimental scheme and assay for repair in T98G (MMEJ) cells arrested at G0/G1 or released to Lov-, Thy-, or Noc-containing medium. (D) U2OS cells with single integration of MMEJ substrate treated ± Rosc for 24 h or expressing Cdk2 shRNAs were assayed.

Fig. 3.

MMEJ is used frequently to repair DSBs in mammalian cells. (A) EGFP-MMEJ/HR-MluI competition repair substrate. (B) U2OS cells with single integration of EGFP-MMEJ/HR-MluI substrate were induced with I-SceI and sorted for EGFP-positive cells, and PCR and digestion analysis was performed with percentages of digestible products shown. (C) U2OS (from B) and BJ and HME1 cell populations carrying EGFP-MMEJ/HR-MluI were assayed as in B, with repair frequencies of three independent experiments. (D and E) EGFP-HR-MluI repair substrate and assay.

Fig. 4.

The role of various repair proteins and Cdk2 in MMEJ and HR. (A and C) U2OS cells with single integration of EGFP-MMEJ/HR-MluI substrate and stably expressing Ku70, Mre11, CtIP, Cdk2, BLM, or Exo1 shRNAs or control were assayed as in Fig. 3. (B and D) U2OS (EGFP-HR) or U2OS (EGFP-MMEJ) cells with BLM, Exo1, or H2AX shRNAs or control were induced with I-SceI and assayed.

Fig. 5.

Recruitment of BLM and Exo1 to laser-induced DSBs depends on Mre11 nuclease activity. U2OS cells stably expressing enhanced blue fluorescent protein-marked Mre11 or CtIP shRNAs or control (A), or HA-Mre11 (WT or H129N) with endogenous Mre11 silenced (B), were transfected with monomeric red fluorescent protein-marked BLM or Exo1. Laser microirradiation, live-cell imaging, and recruitment analyses were performed as described in SI Materials and Methods. (C) A proposed two-step model for the regulation of MMEJ and HR in mammalian cells.

Fig. 6.

MMEJ is used frequently to repair DSBs at collapsed replication forks. (A and B) U2OS cells with single integration of EGFP-HR and EGFP-MMEJ substrate were sorted for EGFP-negative cells and (in A) assayed for EGFP-positive events or (in B) treated with 2 mM HU, recovered for 14 h and assayed. (C) U2OS (EGFP-MMEJ) with ATR- or Tim-shRNAs or control were treated with HU (as in B) and assayed. (D and E) U2OS (EGFP-MMEJ) cells with LigIII shRNA or control were induced with I-SceI (in D) or HU (in E, treated as in C) and assayed. (F) Model for replication restart and DSB repair at collapsed replication forks (Discussion).