Homology and enzymatic requirements of microhomology-dependent alternative end joining.
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ABSTRACT:
SUBMITTER: Sharma S
PROVIDER: S-EPMC4385936 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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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Project description:The human disorder ataxia telangiectasia (AT), which is characterized by genetic instability and neurodegeneration, results from mutation of the ataxia telangiectasia mutated (ATM) kinase. The loss of ATM leads to cell cycle checkpoint deficiencies and other DNA damage signaling defects that do not fully explain all pathologies associated with A-T including neuronal loss. In addressing this enigma, we find here that ATM suppresses DNA double-strand break (DSB) repair by microhomology-mediated end joining (MMEJ). We show that ATM repression of DNA end-degradation is dependent on its kinase activities and that Mre11 is the major nuclease behind increased DNA end-degradation and MMEJ repair in A-T. Assessment of MMEJ by an in vivo reporter assay system reveals decreased levels of MMEJ repair in Mre11-knockdown cells and in cells treated with Mre11-nuclease inhibitor mirin. Structure-based modeling of Mre11 dimer engaging DNA ends suggests the 5' ends of a bridged DSB are juxtaposed such that DNA unwinding and 3'-5' exonuclease activities may collaborate to facilitate simultaneous pairing of extended 5' termini and exonucleolytic degradation of the 3' ends in MMEJ. Together our results provide an integrated understanding of ATM and Mre11 in MMEJ: ATM has a critical regulatory function in controlling DNA end-stability and error-prone DSB repair and Mre11 nuclease plays a major role in initiating MMEJ in mammalian cells. These functions of ATM and Mre11 could be particularly important in neuronal cells, which are post-mitotic and therefore depend on mechanisms other than homologous recombination between sister chromatids to repair DSBs.
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Project description:Microhomology-mediated end joining (MMEJ) anneals short, imperfect microhomologies flanking DNA breaks, producing repair products with deletions in a Ku- and RAD52-independent fashion. Puzzlingly, MMEJ preferentially selects certain microhomologies over others, even when multiple microhomologies are available. To define rules and parameters for microhomology selection, we altered the length, the position, and the level of mismatches to the microhomologies flanking homothallic switching (HO) endonuclease-induced breaks and assessed their effect on MMEJ frequency and the types of repair product formation. We found that microhomology of eight to 20 base pairs carrying no more than 20% mismatches efficiently induced MMEJ. Deletion of MSH6 did not impact MMEJ frequency. MMEJ preferentially chose a microhomology pair that was more proximal from the break. Interestingly, MMEJ events preferentially retained the centromere proximal side of the HO break, while the sequences proximal to the telomere were frequently deleted. The asymmetry in the deletional profile among MMEJ products was reduced when HO was induced on the circular chromosome. The results provide insight into how cells search and select microhomologies for MMEJ in budding yeast.
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Project description:Ku or DNA ligase 4-independent alternative end joining (alt-EJ) repair of DNA double-strand breaks (DSBs) frequently correlates with increased junctional microhomology. However, alt-EJ also produces junctions without microhomology (apparent blunt joins), and the exact role of microhomology in both alt-EJ and classical non-homologous end joining (NHEJ) remains unclear. To better understand the degree to which alt-EJ depends on annealing at pre-existing microhomologies, we examined inaccurate repair of an I-SceI DSB lacking nearby microhomologies of greater than four nucleotides in Drosophila. Lig4 deficiency affected neither frequency nor length of junctional microhomology, but significantly increased insertion frequency. Many insertions appeared to be templated. Based on sequence analysis of repair junctions, we propose a model of synthesis-dependent microhomology-mediated end joining (SD-MMEJ), in which de novo synthesis by an accurate non-processive DNA polymerase creates microhomology. Repair junctions with apparent blunt joins, junctional microhomologies and short indels (deletion with insertion) are often considered to reflect different repair mechanisms. However, a majority of each type had structures consistent with the predictions of our SD-MMEJ model. This suggests that a single underlying mechanism could be responsible for all three repair product types. Genetic analysis indicates that SD-MMEJ is Ku70, Lig4 and Rad51-independent but impaired in mus308 (POLQ) mutants.
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Project description:Squamous cell carcinomas (SCCs) arising from aerodigestive or anogenital epithelium that are associated with the human papillomavirus (HPV) are far more readily cured with radiation therapy than HPV-negative SCCs. The mechanism behind this increased radiosensitivity has been proposed to be secondary to defects in DNA repair, although the specific repair pathways that are disrupted have not been elucidated. To gain insight into this important biomarker of radiosensitivity, we first examined genomic patterns reflective of defects in DNA double-strand break repair, comparing HPV-associated and HPV-negative head and neck cancers (HNSCC). Compared to HPV-negative HNSCC genomes, HPV+ cases demonstrated a marked increase in the proportion of deletions with flanking microhomology, a signature associated with a backup, error-prone double-strand break repair pathway known as microhomology-mediated end-joining (MMEJ). Then, using 3 different methodologies to comprehensively profile double-strand break repair pathways in isogenic paired cell lines, we demonstrate that the HPV16 E7 oncoprotein suppresses canonical nonhomologous end-joining (NHEJ) and promotes error-prone MMEJ, providing a mechanistic rationale for the clinical radiosensitivity of these cancers.
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Project description:Microhomology (MH) flanking a DNA double-strand break (DSB) drives chromosomal rearrangements but its role in mutagenesis has not yet been analyzed. Here we determined the mutation frequency of a URA3 reporter gene placed at multiple locations distal to a DSB, which is flanked by different sizes (15-, 18-, or 203-bp) of direct repeat sequences for efficient repair in budding yeast. Induction of a DSB accumulates mutations in the reporter gene situated up to 14-kb distal to the 15-bp MH, but more modestly to those carrying 18- and 203-bp or no homology. Increased mutagenesis in MH-mediated end joining (MMEJ) appears coupled to its slower repair kinetics and the extensive resection occurring at flanking DNA. Chromosomal translocations via MMEJ also elevate mutagenesis of the flanking DNA sequences 7.1 kb distal to the breakpoint junction as compared to those without MH. The results suggest that MMEJ could destabilize genomes by triggering structural alterations and increasing mutation burden.
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Project description:Proper DNA double-strand break (DSB) repair is essential for maintaining genome integrity. Microhomology-mediated end joining (MMEJ) is an error-prone repair mechanism, which introduces mutations at break sites and contributes to chromosomal translocations and telomere fusions, thus driving carcinogenesis. Mitotic kinases PLK1, CDK1 and Aurora A are important for supporting MMEJ and are often overexpressed in various tumors. However, the functional interplay between these kinases and MMEJ has not been explored. Here, we found that MMEJ is preferentially employed to fix DSBs in cells arrested in mitosis following nocodazole treatment. We further showed that the DSB repair factor CtIP is jointly phosphorylated by CDK1/Aurora A and PLK1. CDK1/Aurora A-mediated CtIP phosphorylation at serine 327 triggers CtIP binding to the PLK1 polo-box domain, which in turn facilitates PLK1 to phosphorylate CtIP mainly at serine 723. A PLK1 phosphor-mimic CtIP mutant fails to initiate extended end resection and is thus unable to mediate homologous recombination and the G2/M checkpoint but can mediate MMEJ. These data imply that PLK1 may target CtIP to promote error-prone MMEJ and inactivate the G2/M checkpoint. These findings have helped elucidate the oncogenic roles of these factors.
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Project description:Alternative non-homologous end joining (alt-NHEJ) was originally identified as a backup repair mechanism in the absence of classical NHEJ (c-NHEJ) factors but recent studies have demonstrated that alt-NHEJ is active even when c-NHEJ as well as homologous recombination is available. The functions of 53BP1 in NHEJ processes are not well understood. Here, we report that 53BP1 promotes DNA double-strand break (DSB) repair and genomic stability not only in c-NHEJ-proficient but also -deficient human G1-phase cells. Using an array of repair substrates we show that these effects of 53BP1 are correlated with a promotion of microhomology-mediated end-joining (MMEJ), a subtype of alt-NHEJ, in G1-phase. Consistent with a specific role in MMEJ we confirm that 53BP1 status does not affect c-NHEJ. 53BP1 supports sequence deletion during MMEJ consistent with a putative role in facilitating end-resection. Interestingly, promotion of MMEJ by 53BP1 in G1-phase cells is only observed in the presence of functional BRCA1. Depletion of both 53BP1 and BRCA1 increases repair needing microhomology usage and augments loss of DNA sequence, suggesting that MMEJ is a highly regulated DSB repair process. Together, these findings significantly expand our understanding of the cell-cycle-dependent roles of 53BP1 in DSB repair.
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Project description:The maize, cut-and-paste transposon Ac/Ds is mobile in Saccharomyces cerevisiae, and DNA sequences of repair products provide strong genetic evidence that hairpin intermediates form in host DNA during this transposition, similar to those formed for V(D)J coding joints in vertebrates. Both DNA strands must be broken for Ac/Ds to excise, suggesting that double-strand break (DSB) repair pathways should be involved in repair of excision sites. In the absence of homologous template, as expected, Ac excisions are repaired by nonhomologous end joining (NHEJ) that can involve microhomologies close to the broken ends. However, unlike repair of endonuclease-induced DSBs, repair of Ac excisions in the presence of homologous template occurs by gene conversion only about half the time, the remainder being NHEJ events. Analysis of transposition in mutant yeast suggests roles for the Mre11/Rad50 complex, SAE2, NEJ1, and the Ku complex in repair of excision sites. Separation-of-function alleles of MRE11 suggest that its endonuclease function is more important in this repair than either its exonuclease or Rad50-binding properties. In addition, the interstrand cross-link repair gene PSO2 plays a role in end joining hairpin ends that is not seen in repair of linearized plasmids and may be involved in positioning transposase cleavage at the transposon ends.
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Project description:One key problem in precision genome editing is the unpredictable plurality of sequence outcomes at the site of targeted DNA double stranded breaks (DSBs). This is due to the typical activation of the versatile Non-homologous End Joining (NHEJ) pathway. Such unpredictability limits the utility of somatic gene editing for applications including gene therapy and functional genomics. For germline editing work, the accurate reproduction of the identical alleles using NHEJ is a labor intensive process. In this study, we propose Microhomology-mediated End Joining (MMEJ) as a viable solution for improving somatic sequence homogeneity in vivo, capable of generating a single predictable allele at high rates (56% ~ 86% of the entire mutant allele pool). Using a combined dataset from zebrafish (Danio rerio) in vivo and human HeLa cell in vitro, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation to prospectively design MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated high levels of mutant allele homogeneity. MMEJ-based DNA repair at these target loci successfully generated F0 mutant zebrafish embryos and larvae that faithfully recapitulated previously reported, recessive, loss-of-function phenotypes. We also tested the generalizability of our approach in cultured human cells. Finally, we provide a novel algorithm, MENTHU (http://genesculpt.org/menthu/), for improved and facile prediction of candidate MMEJ loci. We believe that this MMEJ-centric approach will have a broader impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of knockout mutant allele at base pair resolution via the standard NHEJ-based approach, we demonstrate that F0 founders transmitted the identical MMEJ allele of interest at high rates. Most importantly, the ability to directly dictate the reading frame of an endogenous target will have important implications for gene therapy applications in human genetic diseases.
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