CtIP promotes microhomology-mediated alternative end joining during class-switch recombination

Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 01/2011; 18(1):75-9. DOI: 10.1038/nsmb.1942
Source: PubMed


Immunoglobulin heavy chain (Igh locus) class-switch recombination (CSR) requires targeted introduction of DNA double strand breaks (DSBs) into repetitive 'switch'-region DNA elements in the Igh locus and subsequent ligation between distal DSBs. Both canonical nonhomologous end joining (C-NHEJ) that seals DNA ends with little or no homology and a poorly defined alternative end joining (A-NHEJ, also known as alt-NHEJ) process that requires microhomology ends for ligation have been implicated in CSR. Here, we show that the DNA end-processing factor CtIP is required for microhomology-directed A-NHEJ during CSR. Additionally, we demonstrate that microhomology joins that are enriched upon depletion of the C-NHEJ component Ku70 require CtIP. Finally, we show that CtIP binds to switch-region DNA in a fashion dependent on activation-induced cytidine deaminase. Our results establish CtIP as a bona fide component of microhomology-dependent A-NHEJ and unmask a hitherto unrecognized physiological role of microhomology-mediated end joining in a C-NHEJ-proficient environment.

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    • "The existence of MMEJ was first demonstrated in mammalian cells [9], and MMEJ was long considered a " back-up " mechanism to repair DSBs when NHEJ and other mechanisms fail. However, recent studies indicate that MMEJ occurs even when canonical NHEJ is functional [10] [11]. Several in vitro studies have shown that MMEJ utilizes Ku-independent repair machinery [12] [13] [14] and still exhibits robust activity in DNA-PKc-deficient cells [15] [16]. "
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    ABSTRACT: DNA double-strand break (DSB) repair is of considerable importance for genomic integrity. Homologous recombination (HR) and non-homologous end joining (NHEJ) are considered as two major mechanistically distinct pathways involved in repairing DSBs. In recent years, another DSB repair pathway, namely, microhomology-mediated end joining (MMEJ), has received increasing attention. MMEJ is generally believed to utilize an alternative mechanism to repair DSBs when NHEJ and other mechanisms fail. In this study, we utilized zebrafish as an in vivo model to study DSB repair and demonstrated that efficient MMEJ repair occurred in the zebrafish genome when DSBs were induced using TALEN (transcription activator-like effector nuclease) or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technologies. The wide existence of MMEJ repair events in zebrafish embryos was further demonstrated via the injection of several in vitro-designed exogenous MMEJ reporters. Interestingly, the inhibition of endogenous ligase 4 activity significantly increased MMEJ frequency, and the inhibition of ligase 3 activity severely decreased MMEJ activity. These results suggest that MMEJ in zebrafish is dependent on ligase 3 but independent of ligase 4. This study will enhance our understanding of the mechanisms of MMEJ in vivo and facilitate inducing desirable mutations via DSB-induced repair. Copyright © 2015 Elsevier B.V. All rights reserved.
    Full-text · Article · Aug 2015 · Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
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    • "It processes ssDNA gaps of 2 to 25 nucleotides in length (McVey & Lee, 2008), while ssDNA gaps 430 nucleotides are repaired by ssDNA annealing (SSA). Proteins involved in MMEJ include BLM/MRN, EXO1 or DNA2, FEN1, DNA polymerase b, or m, Ligase I or Ligase III/XRCC1 and MMR proteins (Figure 1) (Crespan et al., 2012; Lee-Theilen et al., 2011; Nimonkar et al., 2011; Paul et al., 2013). After ssDNA is annealed in the gap region, mismatched bases are corrected by MMR. "
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    ABSTRACT: Abstract DNA double-strand breaks are highly toxic DNA lesions that cause genomic instability, if not efficiently repaired. RecQ helicases are a family of highly conserved proteins that maintain genomic stability through their important roles in several DNA repair pathways, including DNA double-strand break repair. Double-strand breaks can be repaired by homologous recombination (HR) using sister chromatids as templates to facilitate precise DNA repair, or by an HR-independent mechanism known as non-homologous end-joining (NHEJ) (error-prone). NHEJ is a non-templated DNA repair process, in which DNA termini are directly ligated. Canonical NHEJ requires DNA-PKcs and Ku70/80, while alternative NHEJ pathways are DNA-PKcs and Ku70/80 independent. This review discusses the role of RecQ helicases in NHEJ, alternative (or back-up) NHEJ (B-NHEJ) and microhomology-mediated end-joining (MMEJ) in V(D)J recombination, class switch recombination and telomere maintenance.
    Full-text · Article · Jul 2014 · Critical Reviews in Biochemistry and Molecular Biology
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    • "Mre11 and CtIP have been implicated to trim broken DNA ends to uncover microhomology regions, generating short stretches of complementary nucleotides at DNA breaks, thereby promoting A-EJ during CSR (116). In CH12F3 cells, CtIP depletion impaired CSR to IgA and reduced the overall length of microhomology at the S junctions (130, 131). Notably, CtIP-deficient B cells undergo normal CSR to IgG1 (132). "
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