The Mechanism of Human Nonhomologous DNA End Joining

Abstract

Double-strand breaks are common in all living cells, and there are two major pathways for their repair. In eukaryotes, homologous recombination is restricted to late S or G2, whereas nonhomologous DNA end joining (NHEJ) can occur throughout the cell cycle and is the major pathway for the repair of double-strand breaks in multicellular eukaryotes. NHEJ is distinctive for the flexibility of the nuclease, polymerase, and ligase activities that are used. This flexibility permits NHEJ to function on the wide range of possible substrate configurations that can arise when double-strand breaks occur, particularly at sites of oxidative damage or ionizing radiation. NHEJ does not return the local DNA to its original sequence, thus accounting for the wide range of end results. Part of this heterogeneity arises from the diversity of the DNA ends, but much of it arises from the many alternative ways in which the nuclease, polymerases, and ligase can act during NHEJ. Physiologic double-strand break processes make use of the imprecision of NHEJ in generating antigen receptor diversity. Pathologically, the imprecision of NHEJ contributes to genome mutations that arise over time. Double-strand breaks are common in all living cells, and there are two major pathways for their repair. In eukaryotes, homologous recombination is restricted to late S or G2, whereas nonhomologous DNA end joining (NHEJ) can occur throughout the cell cycle and is the major pathway for the repair of double-strand breaks in multicellular eukaryotes. NHEJ is distinctive for the flexibility of the nuclease, polymerase, and ligase activities that are used. This flexibility permits NHEJ to function on the wide range of possible substrate configurations that can arise when double-strand breaks occur, particularly at sites of oxidative damage or ionizing radiation. NHEJ does not return the local DNA to its original sequence, thus accounting for the wide range of end results. Part of this heterogeneity arises from the diversity of the DNA ends, but much of it arises from the many alternative ways in which the nuclease, polymerases, and ligase can act during NHEJ. Physiologic double-strand break processes make use of the imprecision of NHEJ in generating antigen receptor diversity. Pathologically, the imprecision of NHEJ contributes to genome mutations that arise over time. nonhomologous DNA end joining double-strand DNA breaks nucleotide(s) DNA-dependent protein kinase catalytic subunit polymerase(s) terminal deoxynucleotidyltransferase microhomology-mediated end joining recombination-activating gene nonhomologous DNA end joining double-strand DNA breaks nucleotide(s) DNA-dependent protein kinase catalytic subunit polymerase(s) terminal deoxynucleotidyltransferase microhomology-mediated end joining recombination-activating gene All living cells have mechanisms for repairing double-strand DNA breaks (DSBs). Pathologic (disadvantageous) DSBs arise when the replication fork encounters a nick. Ionizing radiation particles create clusters of reactive oxygen species along their path, and these create DSBs. Reactive oxygen species themselves may cause DSBs. For dividing mammalian cells in culture, 5–10% appear to have at least one chromosome break (or chromatid gap) at any one time (1Lieber M.R. Karanjawala Z.E. Nat. Rev. Mol. Cell Biol. 2004; 5: 69-75Crossref PubMed Scopus (103) Google Scholar). Hence, the need to repair DSBs arises commonly (2Li H. Vogel H. Holcomb V.B. Gu Y. Hasty P. Mol. Cell. Biol. 2007; 27: 8205-8214Crossref PubMed Scopus (110) Google Scholar). There are two primary pathways for the repair of DSBs (Fig. 1). Homologous recombination occurs in during late S or G2 of the cell cycle when the sister chromatid is close in proximity (3Sonoda E. Hochegger H. Saberi A. Taniguchi Y. Takeda S. DNA Repair. 2006; 5: 1021-1029Crossref PubMed Scopus (386) Google Scholar). NHEJ is the major pathway for the repair of DSBs because it can function throughout the cell cycle and because it does not require a homologous chromosome (3Sonoda E. Hochegger H. Saberi A. Taniguchi Y. Takeda S. DNA Repair. 2006; 5: 1021-1029Crossref PubMed Scopus (386) Google Scholar, 4Moore J.K. Haber J.E. Mol. Cell. Biol. 1996; 16: 2164-2173Crossref PubMed Scopus (599) Google Scholar). Rather, NHEJ involves rejoining of what remains of the two DNA ends, and the mechanism has evolved in a manner that tolerates nucleotide (nt) loss or addition at the rejoining site. Because of the few nucleotides of resection and random addition necessary to get the two DNA ends into a ligatable configuration, NHEJ is distinctive among major DNA repair pathways for its imprecision. Hence, NHEJ leaves “information scars” at most sites of repair in vertebrates. The positive aspect of NHEJ is that the phosphodiester backbone and structural integrity of the chromosome are restored at sites that would otherwise result in loss of several hundreds of genes on entire chromosomal arms or segments. Attendant with the imprecision of NHEJ is the accumulation of randomly located mutations over time in the genome of each somatic cell of an organism. Like most DNA repair processes, there are three enzymatic activities required for repair of DSBs by the NHEJ pathway: (a) nucleases to remove damaged DNA, (b) polymerases to aid in the repair, and (c) a ligase to restore the phosphodiester backbone (Fig. 2). In vertebrates, the Artemis·DNA-dependent protein kinase catalytic subunit (DNA-PKcs) complex becomes active as a 5′- and 3′-endonuclease when DNA-PKcs binds to a DSB DNA end. Polymerases μ and λ are two of the known polymerases for NHEJ. A complex of XLF (Cernunnos), XRCC4, and DNA ligase IV composes the ligase for NHEJ. When a DSB occurs during G0, G1, and early S phase, the Ku heterodimer (Ku70/Ku80) appears likely to be the first protein to bind. (During late S or G2, there may be some competition between NHEJ and homologous recombination, although this is still an active area of investigation (5Adachi N. Ishino T. Ishii Y. Takeda S. Koyama H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12109-12113Crossref PubMed Scopus (123) Google Scholar).) Ku must change conformation once it binds DNA because its interactions with other proteins such as DNA-PKcs are much stronger once the Ku·DNA end complex has formed (6Lieber M.R. Yu K. Raghavan S.C. DNA Repair. 2006; 5: 1234-1245Crossref PubMed Scopus (153) Google Scholar). Ku is capable of interacting with the nuclease (Artemis·DNA-PKcs), the polymerases (μ and λ), and the ligase (XLF·XRCC4·DNA ligase IV). Hence, one can think of Ku as a tool belt protein that can stabilize any of a number of enzymatic activities at a DNA end (Fig. 2). One might assume that the nuclease, polymerases, and ligase function in this order (Fig. 3), and in some of the simpler scenarios, this probably occurs. However, each of these enzymes has a range of flexibility in their function that permits the NHEJ process to go to completion in any of a large number (hundreds) of ways, even when starting with two identical DNA ends. This flexibility accounts for the very diverse number of results, with some ends showing nt loss (1–10 nt, typically) and some joining sites (junctions) showing untemplated nt addition (0–3 nt, typically) (Fig. 2, lower). The next three sections describe in detail the flexibility of the nuclease, polymerase, and ligase components in NHEJ. DNA-PKcs can bind to DNA ends with a KD of 3 × 10–9m, but this affinity improves to 3 × 10–11m at a Ku·DNA end complex (7West R.B. Yaneva M. Lieber M.R. Mol. Cell. Biol. 1998; 18: 5908-5920Crossref PubMed Scopus (145) Google Scholar). Artemis and DNA-PKcs exist as a complex within cells (8Ma Y. Pannicke U. Schwarz K. Lieber M.R. Cell. 2002; 108: 781-794Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar), and this complex binds to Ku·DNA end complexes. When Ku moves internally, this permits DNA-PKcs to contact the DNA end, which then activates the serine/threonine kinase activity of DNA-PKcs (9Hammarsten O. DeFazio L.G. Chu G. J. Biol. Chem. 2000; 275: 1541-1550Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 10Chu G. J. Biol. Chem. 1997; 272: 24097-24100Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 11Yoo S. Dynan W.S. Nucleic Acids Res. 1999; 27: 4679-4686Crossref PubMed Scopus (160) Google Scholar). Activation of the kinase activity represents one of the simplest signal transduction systems because it permits DNA-PKcs to phosphorylate itself and Artemis (8Ma Y. Pannicke U. Schwarz K. Lieber M.R. Cell. 2002; 108: 781-794Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar, 12Meek K. Gupta S. Ramsden D.A. Lees-Miller S.P. Immunol. Rev. 2004; 200: 132-141Crossref PubMed Scopus (179) Google Scholar). The autophosphorylation of DNA-PKcs causes a conformational change in DNA-PKcs that regulates access by other NHEJ proteins (12Meek K. Gupta S. Ramsden D.A. Lees-Miller S.P. Immunol. Rev. 2004; 200: 132-141Crossref PubMed Scopus (179) Google Scholar, 13Uematsu N. Weterings E. Yano K. Morotomi-Yano K. Jakob B. Taucher-Scholz G. Mari P.O. van Gent D.C. Chen B.P. Chen D.J. J. Cell Biol. 2007; 177: 219-229Crossref PubMed Scopus (312) Google Scholar, 14Jovanovic M. Dynan W.S. Nucleic Acids Res. 2006; 34: 1112-1120Crossref PubMed Scopus (30) Google Scholar, 15Goodarzi A.A. Yu Y. Riballo E. Douglas P. Walker S.A. Ye R. Harer C. Marchetti C. Morrice N. Jeggo P.A. Lees-Miller S.P. EMBO J. 2006; 25: 3880-3889Crossref PubMed Scopus (237) Google Scholar). This conformational change in DNA-PKcs may alter the conformation of Artemis because now Artemis can function as a 5′- or 3′-endonuclease at overhangs (supplemental Fig. 1) (8Ma Y. Pannicke U. Schwarz K. Lieber M.R. Cell. 2002; 108: 781-794Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar, 16Niewolik D. Pannicke U. Lu H. Ma Y. Wang L.C. Kulesza P. Zandi E. Lieber M.R. Schwarz K. J. Biol. Chem. 2006; 281: 33900-33909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). This conformational change in Artemis also permits it to function as an endonuclease at a variety of other single/double-strand DNA structures, including DNA hairpins (8Ma Y. Pannicke U. Schwarz K. Lieber M.R. Cell. 2002; 108: 781-794Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar, 17Ma Y. Schwarz K. Lieber M.R. DNA Repair. 2005; 4: 845-851Crossref PubMed Scopus (134) Google Scholar), which turns out to be critical in the gene rearrangement process of the vertebrate immune system (called V(D)J recombination). One DNA-PKcs molecule can autophosphorylate itself in cis or another DNA-PKcs molecule in trans, and the relative ratio of cis- versus trans-phosphorylation is not known (supplemental Fig. 2) (18Meek K. Douglas P. Cui X. Ding Q. Lees-Miller S.P. Mol. Cell. Biol. 2007; 27: 3881-3890Crossref PubMed Scopus (139) Google Scholar). A subset of DSBs created by ionizing radiation cannot be repaired without Artemis (19Riballo E. Kuhne M. Rief N. Doherty A. Smith G.C. Recio M.J. Reis C. Dahm K. Fricke A. Krempler A. Parker A.R. Jackson S.P. Gennery A. Jeggo P.A. Lobrich M. Mol. Cell. 2004; 16: 715-724Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar). Genetic and biochemical evidence for the role of pol X polymerases exists in yeast and mammalian cells (20Wilson T.E. Lieber M.R. J. Biol. Chem. 1999; 274: 23599-23609Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 21Bertocci B. DeSmet A. Weill J-C. Reynaud C.A. Immunity. 2006; 25: 31-41Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). In yeast, Pol4 is the only pol X polymerase. In mammalian cells, the pol X family consists of pol β, μ, and λ and terminal deoxynucleotidyltransferase (TdT). The latter three all contain BRCT domains, and all three function in NHEJ, whereas pol β does not. pol μ and λ share the most homology with Saccharomyces cerevisiae Pol4, and both appear to be expressed in mammalian somatic cells. TdT is expressed only in pre-B and pre-T cells. pol λ, pol μ, and TdT share a range of structural similarities that correlate with their ratio of template-dependent versus template-independent synthesis (22Moon A.F. Garcia-Diaz M. Batra V.K. Beard W.A. Bebenek K. Kunkel T.A. Wilson S.H. Pedersen L.C. DNA Repair. 2007; 6: 1709-1725Crossref PubMed Scopus (152) Google Scholar). TdT carries out template-independent synthesis, and pol λ is almost exclusively template-dependent. However, polμcan carryout both template-dependent and template-independent synthesis in the presence of the physiologic divalent cation Mg2+ (23Gu J. Lu H. Tippin B. Shimazaki N. Goodman M.F. Lieber M.R. EMBO J. 2007; 26: 1010-1023Crossref PubMed Scopus (125) Google Scholar). pol μ and, to a greater extent, pol λ show some degree of template slippage, which can result in the generation of direct repeats, a feature seen at some sites of NHEJ (supplemental Fig. 3). The template-independent addition by pol μ could conceivably result in fold-back at the region of addition, followed by synthesis using the same strand as a template (supplemental Fig. 3, lower reaction). This could result in the generation of inverted repeats. The generation of direct and inverted repeats at a subset of NHEJ events in vivo is called T-nucleotide addition, where “T” stands for “templated” (24Jaeger U. Bocskor S. Le T. Mitterbauer G. Bolz I. Chott A. Kneba A. Mannhalter C. Nadel B. Blood. 2000; 95: 3520-3529Crossref PubMed Google Scholar). The flexibility of pol μ and λ might account for T-nucleotides. TdT functions only during V(D)J recombination to add random nucleotides (called N-nucleotides) so as to increase the junctional diversity during the generation of the antigen receptor repertoire. The template-independent synthesis by pol μ is the likely basis for occasional antigen receptor junctional additions in lymphocytes from animals in which TdT has been knocked out (25Gilfillan S. Dierich A. Lemeur M. Benoist C. Mathis D. Science. 1993; 261: 1755-1759Crossref PubMed Scopus (376) Google Scholar, 26Komori T. Okada A. Stewart V. Alt F. Science. 1993; 261: 1171-1175Crossref PubMed Scopus (399) Google Scholar). One might wonder why pol μ, a polymerase present in all somatic cells, would add nucleotides randomly. The evolutionary advantage of this becomes clear when considering a DSB in which there is no terminal microhomology between the two DNA ends. Random addition has the benefit of potentially generating 1 or 2 nt of terminal microhomology, permitting more efficient annealing of the ends and thereby facilitating NHEJ. Hence, use of terminal microhomology occurs in some fraction of NHEJ events when it exists between the two ends, and pol μ may manufacture such terminal microhomology when it does not exist at the two DNA ends. In addition, when 3′-overhangs are involved, pol μ may be more robust at this type of fill-in from a region of minimal end-to-end annealing than any other polymerase (27Daley J.M. Vander Laan R.L. Suresh A. Wilson T.E. J. Biol. Chem. 2005; 280: 29030-29037Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 28Daley J.M. Palmbos P.L. Wu D. Wilson T.E. 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All of these of the flexibility to the range of DNA end configurations that XRCC4·DNA ligase IV can in its role as the only ligase for the repair of DSBs. that the two DNA ends of the DSB are likely to be in close each DNA end is likely to have a Ku molecule and with any of the three enzymatic permitting nuclease, polymerase, and ligase (supplemental and damage sites to ionizing nt have been at the of loss of a few more nt during the NHEJ repair process is of this is the with the of not the chromosomal of some of imprecision has likely the nuclease, polymerases, and ligase of NHEJ to the substrate and catalytic flexibility that have advantage of the imprecision and flexibility of NHEJ in the generation of antigen for the of the immune In V(D)J recombination, the imprecision at the and joining sites (junctions) the of diversity that would otherwise be to the of and segments. has that many have an NHEJ system that may be simpler than the one in eukaryotes, but which the same function F. 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However, the other NHEJ components are still and in which NHEJ cannot go to enzymes from other pathways might be to to the that their enzymatic For in a ligase the polymerase activities are to for to the strand from the strand DNA between the then ligase or would be to the of the of an NHEJ this use of another ligase might be very The of these or to joining relative to NHEJ is not these not appear capable of at the of NHEJ in to ionizing radiation or V(D)J recombination out by proteins B. Cui X. I. R. S. S. G. B. V. K. 2007; PubMed Scopus Google Scholar). In recombination, the are in repeats that may the overhangs at the two DNA ends to be with polymerase, and by ligase or in of ligase IV C. S. M. S. M. A.A. K. Alt 2007; PubMed Scopus Google Scholar). The that these repeats are not for joining that NHEJ is for the large of end joining in The most commonly alternative end joining pathway to NHEJ is called microhomology-mediated end joining which is a that a subset of NHEJ events also nt of terminal microhomology Haber J.E. Mol. Cell. Biol. PubMed Scopus Google Scholar). terminal microhomology microhomology is in and the of the microhomology are than in NHEJ. The ligase for a subset of events is ligase but that for is ligase IV in yeast Haber J.E. Mol. Cell. Biol. PubMed Scopus Google Scholar). In mammalian cells, there is evidence for ligase H. B. R. Wang M. F. G. Res. 2005; PubMed Scopus Google Scholar), that and other alternative to may the that can arise when the of NHEJ components is not but that are not of physiologic This likely that the of DSBs that arise such as with ionizing radiation and oxidative would almost the of DNA ends that are required for or many other alternative ends that share several nt of terminal The ligase joining to in cells F. Wu Wang M. J. B. G. J. Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar), which is the of what would for in an end joining in cells for NHEJ, other enzymes may but with a of of and than such events it would be to these events ligase NHEJ or NHEJ, NHEJ early in and is present in many and all eukaryotes. NHEJ is at the local but efficient at chromosomal structural The enzymatic of NHEJ are but still a of a it is not clear what are required to NHEJ and these are between the completion of the DSB repair and the cell cycle is another area for DNA it is not clear what a DSB is repaired by NHEJ versus homologous there is still much to be this DNA repair K. D. and X. Cui for on the with