Microhomology-mediated end joining: new players join the team
© The Author(s) 2017
Received: 25 December 2016
Accepted: 6 January 2017
Published: 13 January 2017
DNA double-strand breaks (DSBs) are the most deleterious type of DNA damage in cells arising from endogenous and exogenous attacks on the genomic DNA. Timely and properly repair of DSBs is important for genomic integrity and survival. MMEJ is an error-prone repair mechanism for DSBs, which relies on exposed microhomologous sequence flanking broken junction to fix DSBs in a Ku- and ligase IV-independent manner. Recently, significant progress has been made in MMEJ mechanism study. In this review, we will summarize its biochemical activities of several newly identified MMEJ factors and their biological significance.
Double-strand breaks (DSBs) are potentially lethal lesions that arise from endogenous and exogenous genotoxic agents [1, 2]. Unrepaired DSBs cause chromosome breaks and translocations that are associated with developmental defects, neurodegeneration, immunodeficiency, radiosensitivity, sterility, and cancer predisposition [3–5]. Non-homologous end joining (NHEJ) and homologous recombination (HR)-mediated DSB repair are two major pathways to fix DSBs [6, 7]. HR is generally considered to be an error-free mechanism because the identical sister chromatids are used as templates to repair DSBs when cells reside at the S and G2 phases. Ku-dependent classical non-homologous end joining (C-NHEJ) is active in all phases of the cell cycle, which can be high fidelity or associate with small alterations at junction since direct end ligation is catalyzed by DNA ligase IV [8–10]. In the absence of Ku protein or in C-NHEJ-deficient cells, resection machinery will expose extensive single strand DNA (ssDNA) which allows cells to use alternative end join (A-NHEJ) or HR as repair mechanism. A subset of A-NHEJ relies on microhomologous sequences on either side of the DSB, thus is named as microhomology-mediated end joining (MMEJ) [10–12]. MMEJ is a mutagenic DSB repair mechanism, which always associates with deletions flanking the break sites and contributes to chromosome translocations and rearrangements. Recent study indicated that MMEJ is used with appreciable frequency even when HR is available . It seems that MMEJ is a crucial DSB repair mechanism for HR-defective tumors . These raised the possibility that MMEJ may not just is a back-up repair mechanism. The molecular mechanism of MMEJ thus draws much attention in the field. Several important MMEJ factors have been identified recently [14–17]. Here, we will discuss biochemical properties and regulatory mechanism of these pivotal factors in MMEJ repair.
Basic mechanisms of MMEJ
Resection factors: mechanisms are still missing
In principle, both HR and MMEJ are initiated by 5′–3′ resection of DSB ends to expose ssDNA overhangs. While HR needs a long 3′-ssDNA tail to invade homologous template, MMEJ requires exposure of two microhomologous regions to anneal each other. Studies in yeast and mammalian cells indicated that DSB end resection may be carried out in two steps: Mre11 complex and Sae2/CtIP remove covalent adducts, such as bound proteins and hairpin-capped ends and initiate end resection. Sgs1/Exo1 and DNA2 in yeast or BLM (human homologue of Sgs1) and Exo1 in human cells take over to produce extended 3′-ssDNA tail [23–28]. It has been demonstrated that both Mre11 and CtIP are important for MMEJ. However, depletion of long-range resection factors including BLM/Exo1 in mammalian cells and Sgs1/Exo1 in yeast significantly increased frequency of MMEJ when the microhomologous regions close to the break site [13, 16, 29]. Possibly, down-regulation of long-range end resection may cause accumulation of short 3’tail containing DSBs which cannot be channeled to HR repair but is sufficient for exposing microhomologous region nearby DSB site and mediating MMEJ. However, we cannot rule out other possibilities yet. For example, some resection factors may harbor multiple functions. Further, the contradictory results have been obtained in studies of BRCA1, which also is a classical DSB end resection factor. BRCA1 closely associates with MRN complex and CtIP. CDK phosphorylation-mediated interaction between CtIP and BRCA1 enhances the speed of CtIP-mediated end resection . Cell cycle dependent BRCA1-MRN-CtIP complex formation has been reported to play a critical role in DSB end resection and HR-mediated DSB repair in mammalian cells . Early work in DT40 (chicken) B cells suggested that MMEJ is not affected by BRCA1 . While, using different human cells, a recent study indicated BRCA1 may work downstream of Mre11 and CtIP to suppress MMEJ . However, in MEFs cells whose telomeres were artificially uncapped, Madalena Tarsounas’s group demonstrated that CtIP and BRCA1 promote MMEJ at uncapped telomeres . Obviously, more accurate systems are needed to clarify the underlining mechanism for the functional relationship between resection factors and MMEJ.
RPA: an old soldier joined new team
Polθ: new focus
Increasing evidences suggest that MMEJ may not just be a back-up DSB repair mechanism. MMEJ occurs even when HR and NHEJ are intact and is essential for HR-deficient cancer cells. Therefore, it is well deserved to fully decipher the molecular mechanisms of MMEJ and its unique function in DSB repair. So far, several key factors identified in both MMEJ repair and regulation have overlapping functions with other repair pathways. Discovery of specific enzymes or protein factors that solely work in MMEJ repair pathway will help us understand the detail mechanism of MMEJ and its unique role in DSB repair and be instrumental for MMEJ-targeted drug design.
HW and XX planned and critically revised the manuscript. Both authors read and approved the final manuscript.
We thank the members of Xu and Wang lab for helpful discussions. We apologize for that we were not able to cite all the works of our colleagues in this review due to space limitation.
The authors declare that they have no competing interests.
Research in the Hailong Wang’s group and Xingzhi Xu’s group is supported by the 973 projects 2015CB910601/2 and 2013CB911002; the National Natural Science Foundation of China (NSFC) Grants 31370841, 31530016, and 31461143012; The Importation and Development of High-Caliber Talents Project of Beijing Municipal Institutions (CIT&TCD201504069).
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- Bassing CH, Alt FW. The cellular response to general and programmed DNA double strand breaks. DNA Repair. 2004;3:781–96.View ArticlePubMedGoogle Scholar
- Khanna KK, Jackson SP. DNA double-strand breaks signaling, repair and the cancer connection. Nat Genet. 2001;27:247–54.View ArticlePubMedGoogle Scholar
- Varon R, Vissinga C, Platzer M, Cerosaletti KM, Chrzanowska KH, Saar K, Beckmann G, Seemanova E, Cooper PR, Nowak NJ, et al. Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome. Cell. 1998;93:467–76.View ArticlePubMedGoogle Scholar
- Stewart GS, Maser RS, Stankovic T, Bressan DA, Kaplan MI, Jaspers NG, Raams A, Byrd PJ, Petrini JH, Taylor AM. The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. Cell. 1999;99:577–87.View ArticlePubMedGoogle Scholar
- Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu Q, Cochran C, Bennett LM, Ding W, et al. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science. 1994;266:66–71.View ArticlePubMedGoogle Scholar
- Lieber MR. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem. 2010;79:181–211.View ArticlePubMedPubMed CentralGoogle Scholar
- Moynahan ME, Jasin M. Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat Rev Mol Cell Biol. 2010;11:196–207.View ArticlePubMedPubMed CentralGoogle Scholar
- Symington LS, Gautier J. Double-strand break end resection and repair pathway choice. Annu Rev Genet. 2011;45:247–71.View ArticlePubMedGoogle Scholar
- Lin WY, Wilson JH, Lin Y. Repair of chromosomal double-strand breaks by precise ligation in human cells. DNA Repair. 2013;12:480–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Chiruvella KK, Liang Z, Wilson TE. Repair of double-strand breaks by end joining. Cold Spring Harb Perspec Biol. 2013;5:a012757.View ArticleGoogle Scholar
- Boulton SJ, Jackson SP. Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways. EMBO J. 1996;15:5093–103.PubMedPubMed CentralGoogle Scholar
- Ma JL, Kim EM, Haber JE, Lee SE. Yeast Mre11 and Rad1 proteins define a Ku-independent mechanism to repair double-strand breaks lacking overlapping end sequences. Mol Cell Biol. 2003;23:8820–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Truong LN, Li Y, Shi LZ, Hwang PY, He J, Wang H, Razavian N, Berns MW, Wu X. Microhomology-mediated end joining and homologous recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells. Proc Natl Acad Sci USA. 2013;110:7720–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Ceccaldi R, Liu JC, Amunugama R, Hajdu I, Primack B, Petalcorin MI, O’Connor KW, Konstantinopoulos PA, Elledge SJ, Boulton SJ, et al. Homologous-recombination-deficient tumours are dependent on Poltheta-mediated repair. Nature. 2015;518:258–62.View ArticlePubMedPubMed CentralGoogle Scholar
- Mateos-Gomez PA, Gong F, Nair N, Miller KM, Lazzerini-Denchi E, Sfeir A. Mammalian polymerase theta promotes alternative NHEJ and suppresses recombination. Nature. 2015;518:254–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Deng SK, Gibb B, de Almeida MJ, Greene EC, Symington LS. RPA antagonizes microhomology-mediated repair of DNA double-strand breaks. Nat Struct Mol Biol. 2014;21:405–12.View ArticlePubMedPubMed CentralGoogle Scholar
- Sfeir A, Symington LS. Microhomology-mediated end joining. A back-up survival mechanism or dedicated pathway? Trends Biochem Sci. 2015;40:701–14.View ArticlePubMedPubMed CentralGoogle Scholar
- Decottignies A. Alternative end-joining mechanisms: a historical perspective. Front Genet. 2013;4:48.View ArticlePubMedPubMed CentralGoogle Scholar
- McVey M, Lee SE. MMEJ repair of double-strand breaks (director’s cut): deleted sequences and alternative endings. Trends Genet. 2008;24:529–38.View ArticlePubMedGoogle Scholar
- Pannunzio NR, Li S, Watanabe G, Lieber MR. Non-homologous end joining often uses microhomology: implications for alternative end joining. DNA Repair (Amst). 2014;17:74–80.View ArticleGoogle Scholar
- Sinha S, Villarreal D, Shim EY, Lee SE. Risky business. Microhomology-mediated end joining. Mutat Res. 2016;788:17–24.View ArticlePubMedGoogle Scholar
- Villarreal DD, Lee K, Deem A, Shim EY, Malkova A, Lee SE. Microhomology directs diverse DNA break repair pathways and chromosomal translocations. PLoS Genet. 2012;8:e1003026.View ArticlePubMedPubMed CentralGoogle Scholar
- Symington LS. End resection at double-strand breaks: mechanism and regulation. Cold Spring Harb Perspect Biol. 2014;6:8.View ArticleGoogle Scholar
- Cejka P, Cannavo E, Polaczek P, Masuda-Sasa T, Pokharel S, Campbell JL, Kowalczykowski SC. DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2. Nature. 2010;467:112–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Niu H, Chung WH, Zhu Z, Kwon Y, Zhao W, Chi P, Prakash R, Seong C, Liu D, Lu L, et al. Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae. Nature. 2010;467:108–11.View ArticlePubMedPubMed CentralGoogle Scholar
- Nimonkar AV, Genschel J, Kinoshita E, Polaczek P, Campbell JL, Wyman C, Modrich P, Kowalczykowski SC. BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair. Genes Dev. 2011;25:350–62.View ArticlePubMedPubMed CentralGoogle Scholar
- Makharashvili N, Tubbs AT, Yang SH, Wang H, Barton O, Zhou Y, Deshpande RA, Lee JH, Lobrich M, Sleckman BP, et al. Catalytic and noncatalytic roles of the CtIP endonuclease in double-strand break end resection. Mol Cell. 2014;54:1022–33.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang H, Li Y, Truong LN, Shi LZ, Hwang PY, He J, Do J, Cho MJ, Li H, Negrete A, et al. CtIP maintains stability at common fragile sites and inverted repeats by end resection-independent endonuclease activity. Mol Cell. 2014;54:1012–21.View ArticlePubMedPubMed CentralGoogle Scholar
- Ahrabi S, Sarkar S, Pfister SX, Pirovano G, Higgins GS, Porter AC, Humphrey TC. A role for human homologous recombination factors in suppressing microhomology-mediated end joining. Nucleic Acids Res. 2016;44:5743–57.View ArticlePubMedPubMed CentralGoogle Scholar
- Cruz-Garcia A, Lopez-Saavedra A, Huertas P. BRCA1 accelerates CtIP-mediated DNA-end resection. Cell Rep. 2014;9:451–9.View ArticlePubMedGoogle Scholar
- Chen L, Nievera CJ, Lee AY, Wu X. Cell cycle-dependent complex formation of BRCA1.CtIP.MRN is important for DNA double-strand break repair. J Biol Chem. 2008;283:7713–20.View ArticlePubMedGoogle Scholar
- Yun MH, Hiom K. CtIP-BRCA1 modulates the choice of DNA double-strand-break repair pathway throughout the cell cycle. Nature. 2009;459:460–3.View ArticlePubMedPubMed CentralGoogle Scholar
- Badie S, Carlos AR, Folio C, Okamoto K, Bouwman P, Jonkers J, Tarsounas M. BRCA1 and CtIP promote alternative non-homologous end-joining at uncapped telomeres. EMBO J. 2015;34:828.View ArticlePubMedPubMed CentralGoogle Scholar
- Iftode C, Daniely Y, Borowiec JA. Replication protein A (RPA): the eukaryotic SSB. Crit Rev Biochem Mol Biol. 1999;34:141–80.View ArticlePubMedGoogle Scholar
- Lao Y, Lee CG, Wold MS. Replication protein A interactions with DNA. 2. Characterization of double-stranded DNA-binding/helix-destabilization activities and the role of the zinc-finger domain in DNA interactions. Biochemistry. 1999;38:3974–84.View ArticlePubMedGoogle Scholar
- Walther AP, Gomes XV, Lao Y, Lee CG, Wold MS. Replication protein A interactions with DNA. 1. Functions of the DNA-binding and zinc-finger domains of the 70-kDa subunit. Biochemistry. 1999;38:3963–73.View ArticlePubMedGoogle Scholar
- Georgaki A, Strack B, Podust V, Hubscher U. DNA unwinding activity of replication protein A. F FEBS Lett. 1992;308:240–4.View ArticleGoogle Scholar
- Zou Y, Liu Y, Wu X, Shell SM. Functions of human replication protein A (RPA): from DNA replication to DNA damage and stress responses. J Cell Physiol. 2006;208:267–73.View ArticlePubMedPubMed CentralGoogle Scholar
- San Filippo J, Sung P, Klein H. Mechanism of eukaryotic homologous recombination. Annu Rev Biochem. 2008;77:229–57.View ArticlePubMedGoogle Scholar
- Mimitou EP, Symington LS. Nucleases and helicases take center stage in homologous recombination. Trends Biochem Sci. 2009;34:264–72.View ArticlePubMedGoogle Scholar
- Seki M, Marini F, Wood RD. POLQ (Pol theta), a DNA polymerase and DNA-dependent ATPase in human cells. Nucleic Acids Res. 2003;31:6117–26.View ArticlePubMedPubMed CentralGoogle Scholar
- Hogg M, Seki M, Wood RD, Doublie S, Wallace SS. Lesion bypass activity of DNA polymerase theta (POLQ) is an intrinsic property of the pol domain and depends on unique sequence inserts. J Mol Biol. 2011;405:642–52.View ArticlePubMedGoogle Scholar
- Arana ME, Seki M, Wood RD, Rogozin IB, Kunkel TA. Low-fidelity DNA synthesis by human DNA polymerase theta. Nucleic Acids Res. 2008;36:3847–56.View ArticlePubMedPubMed CentralGoogle Scholar
- Harris PV, Mazina OM, Leonhardt EA, Case RB, Boyd JB, Burtis KC. Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes. Mol Cell Biol. 1996;16:5764–71.View ArticlePubMedPubMed CentralGoogle Scholar
- Marini F, Wood RD. A human DNA helicase homologous to the DNA cross-link sensitivity protein Mus308. J Biol Chem. 2002;277:8716–23.View ArticlePubMedGoogle Scholar
- Boyd JB, Sakaguchi K, Harris PV. mus308 mutants of Drosophila exhibit hypersensitivity to DNA cross-linking agents and are defective in a deoxyribonuclease. Genetics. 1990;125:813–9.PubMedPubMed CentralGoogle Scholar
- Muzzini DM, Plevani P, Boulton SJ, Cassata G, Marini F. Caenorhabditis elegans POLQ-1 and HEL-308 function in two distinct DNA interstrand cross-link repair pathways. DNA Repair (Amst). 2008;7:941–50.View ArticlePubMedGoogle Scholar
- Zan H, Shima N, Xu Z, Al-Qahtani A, Evinger Iii AJ, Zhong Y, Schimenti JC, Casali P. The translesion DNA polymerase theta plays a dominant role in immunoglobulin gene somatic hypermutation. EMBO J. 2005;24:3757–69.View ArticlePubMedPubMed CentralGoogle Scholar
- Masuda K, Ouchida R, Takeuchi A, Saito T, Koseki H, Kawamura K, Tagawa M, Tokuhisa T, Azuma T. J OW. DNA polymerase theta contributes to the generation of C/G mutations during somatic hypermutation of Ig genes. Proc Natl Acad Sci USA. 2005;102:13986–91.View ArticlePubMedPubMed CentralGoogle Scholar
- Yoshimura M, Kohzaki M, Nakamura J, Asagoshi K, Sonoda E, Hou E, Prasad R, Wilson SH, Tano K, Yasui A, et al. Vertebrate POLQ and POLbeta cooperate in base excision repair of oxidative DNA damage. Mol Cell. 2006;24:115–25.View ArticlePubMedPubMed CentralGoogle Scholar
- Chan SH, Yu AM, McVey M. Dual roles for DNA polymerase theta in alternative end-joining repair of double-strand breaks in Drosophila. PLoS Genet. 2010;6:e1001005.View ArticlePubMedPubMed CentralGoogle Scholar
- Yu AM, McVey M. Synthesis-dependent microhomology-mediated end joining accounts for multiple types of repair junctions. Nucleic Acids Res. 2010;38:5706–17.View ArticlePubMedPubMed CentralGoogle Scholar
- Kent T, Chandramouly G, McDevitt SM, Ozdemir AY, Pomerantz RT. Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase theta. Nat Struct Mol Biol. 2015;22:230–7.View ArticlePubMedPubMed CentralGoogle Scholar