APLF promotes the assembly and activity of non‐homologous end joining protein complexes

Non‐homologous end joining (NHEJ) is critical for the maintenance of genetic integrity and DNA double‐strand break (DSB) repair. NHEJ is regulated by a series of interactions between core components of the pathway, including Ku heterodimer, XLF/Cernunnos, and XRCC4/DNA Ligase 4 (Lig4). However, the mechanisms by which these proteins assemble into functional protein–DNA complexes are not fully understood. Here, we show that the von Willebrand (vWA) domain of Ku80 fulfills a critical role in this process by recruiting Aprataxin‐and‐PNK‐Like Factor (APLF) into Ku‐DNA complexes. APLF, in turn, functions as a scaffold protein and promotes the recruitment and/or retention of XRCC4‐Lig4 and XLF, thereby assembling multi‐protein Ku complexes capable of efficient DNA ligation in vitro and in cells. Disruption of the interactions between APLF and either Ku80 or XRCC4‐Lig4 disrupts the assembly and activity of Ku complexes, and confers cellular hypersensitivity and reduced rates of chromosomal DSB repair in avian and human cells, respectively. Collectively, these data identify a role for the vWA domain of Ku80 and a molecular mechanism by which DNA ligase proficient complexes are assembled during NHEJ in mammalian cells, and reveal APLF to be a structural component of this critical DSB repair pathway.     

Genistein-induced DNA damage is repaired by nonhomologous end joining and homologous recombination in TK6 cells

Genistein (GES), a phytoestrogen, has potential chemopreventive and chemotherapeutic effects on cancer. The anticancer mechanism of GES may be related with topoisomerase II associated DNA double-strand breaks (DSBs). However, the precise molecular mechanism remains elusive. Here, we performed genetic analyses using human lymphoblastoid TK6 cell lines to investigate whether non-homologous DNA end joining (NHEJ) and homologous recombination (HR), the two major repair pathways of DSBs, were involved in repairing GES-induced DNA damage. Our results showed that GES induced DSBs in TK6 cells. Cells lacking Ligase4, an NHEJ enzyme, are hypersensitive to GES. Furthermore, the sensitivity of Ligase4^−/− cells was associated with enhanced DNA damage when comparing the accumulation of γ-H2AX foci and number of chromosomal aberrations (CAs) with WT cells. In addition, cells lacking Rad54, a HR enzyme, also presented hypersensitivity and increased DNA damages in response to GES. Meanwhile, Treatment of GES-lacking enhanced the accumulation of Rad51, an HR factor, in TK6 cells, especially in Ligase4^−/−. These results provided direct evidence that GES induced DSBs in TK6 cells and clarified that both NHEJ and HR were involved in the repair of GES-induced DNA damage, suggesting that GES in combination with inhibition of NHEJ or HR would provide a potential anticancer strategy.     

Agrobacterium T‐DNA integration into the plant genome can occur without the activity of key non‐homologous end‐joining proteins

Non‐homologous end joining (NHEJ) is the major model proposed for Agrobacterium T‐DNA integration into the plant genome. In animal cells, several proteins, including KU70, KU80, ARTEMIS, DNA‐PKcs, DNA ligase IV (LIG4), Ataxia telangiectasia mutated (ATM), and ATM‐ and Rad3‐related (ATR), play an important role in ‘classical’ (c)NHEJ. Other proteins, including histone H1 (HON1), XRCC1, and PARP1, participate in a ‘backup’ (b)NHEJ process. We examined transient and stable transformation frequencies of Arabidopsis thaliana roots mutant for numerous NHEJ and other related genes. Mutants of KU70, KU80, and the plant‐specific DNA LIGASE VI (LIG6) showed increased stable transformation susceptibility. However, these mutants showed transient transformation susceptibility similar to that of wild‐type plants, suggesting enhanced T‐DNA integration in these mutants. These results were confirmed using a promoter‐trap transformation vector that requires T‐DNA integration into the plant genome to activate a promoterless gusA (uidA) gene, by virus‐induced gene silencing (VIGS) of Nicotiana benthamiana NHEJ genes, and by biochemical assays for T‐DNA integration. No alteration in transient or stable transformation frequencies was detected with atm, atr, lig4, xrcc1, or parp1 mutants. However, mutation of parp1 caused high levels of T‐DNA integration and transgene methylation. A double mutant (ku80/parp1), knocking out components of both NHEJ pathways, did not show any decrease in stable transformation or T‐DNA integration. Thus, T‐DNA integration does not require known NHEJ proteins, suggesting an alternative route for integration.     

Structural mechanism of ATP‐dependent DNA binding and DNA end bridging by eukaryotic Rad50

The Mre11–Rad50–Nbs1 (MRN) complex is a central factor in the repair of DNA double‐strand breaks (DSBs). The ATP‐dependent mechanisms of how MRN detects and endonucleolytically processes DNA ends for the repair by microhomology‐mediated end‐joining or further resection in homologous recombination are still unclear. Here, we report the crystal structures of the ATPγS‐bound dimer of the Rad50^NBD (nucleotide‐binding domain) from the thermophilic eukaryote Chaetomium thermophilum (Ct) in complex with either DNA or CtMre11^RBD (Rad50‐binding domain) along with small‐angle X‐ray scattering and cross‐linking studies. The structure and DNA binding motifs were validated by DNA binding experiments in vitro and mutational analyses in Saccharomyces cerevisiae in vivo. Our analyses provide a structural framework for the architecture of the eukaryotic Mre11–Rad50 complex. They show that a Rad50 dimer binds approximately 18 base pairs of DNA along the dimer interface in an ATP‐dependent fashion or bridges two DNA ends with a preference for 3′ overhangs. Finally, our results may provide a general framework for the interaction of ABC ATPase domains of the Rad50/SMC/RecN protein family with DNA.     

SHLD2/FAM35A co‐operates with REV7 to coordinate DNA double‐strand break repair pathway choice

DNA double‐strand breaks (DSBs) can be repaired by two major pathways: non‐homologous end‐joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)‐based approach, we identify 11 high‐confidence REV7 interactors and elucidate the role of SHLD2 (previously annotated as FAM35A and RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ‐mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B cells. FAM35A accumulates at DSBs in a 53BP1‐, RIF1‐, and REV7‐dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and SHLD1 (previously annotated as C20orf196 and RINN3), which promotes NHEJ and limits HR. Together, these results establish SHLD2 as a novel effector of REV7 in controlling the decision‐making process during DSB repair.     

TRF2/RAP1 and DNA–PK mediate a double protection against joining at telomeric ends

DNA-dependent protein kinase (DNA-PK) is a double-strand breaks repair complex, the subunits of which (KU and DNA-PKcs) are paradoxically present at mammalian telomeres. Telomere fusion has been reported in cells lacking these proteins, raising two questions: how is DNA–PK prevented from initiating classical ligase IV (LIG4)-dependent non-homologous end-joining (C-NHEJ) at telomeres and how is the backup end-joining (EJ) activity (B-NHEJ) that operates at telomeres under conditions of C-NHEJ deficiency controlled? To address these questions, we have investigated EJ using plasmid substrates bearing double-stranded telomeric tracks and human cell extracts with variable C-NHEJ or B-NHEJ activity. We found that (1) TRF2/RAP1 prevents C-NHEJ-mediated end fusion at the initial DNA–PK end binding and activation step and (2) DNA–PK counteracts a potent LIG4-independent EJ mechanism. Thus, telomeres are protected against EJ by a lock with two bolts. These results account for observations with mammalian models and underline the importance of alternative non-classical EJ pathways for telomere fusions in cells.     

ATM and Artemis promote homologous recombination of radiation‐induced DNA double‐strand breaks in G2

Homologous recombination (HR) and non‐homologous end joining (NHEJ) represent distinct pathways for repairing DNA double‐strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ‐dependent process, which repairs a defined subset of radiation‐induced DSBs in G1‐phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB‐repair pathway whereas HR is only essential for repair of ～15% of X‐ or γ‐ray‐induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation‐induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP‐1, providing evidence that HR in G2 repairs heterochromatin‐associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single‐stranded DNA and Rad51 foci at radiation‐induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ.     

Distinct DNA repair pathways involving RecA and nonhomologous end joining in Mycobacterium smegmatis

Mycobacterium smegmatis was used to study the relationship between DNA repair processes involving RecA and nonhomologous end joining (NHEJ). The effect of gene deletions in recA and/or in two genes involved in NHEJ (ku and ligD) was tested on the ability of bacteria to join breaks in plasmids transformed into them and in their response to chemicals that damage DNA. The results provide in vivo evidence that only NHEJ is required for the repair of noncompatible DNA ends. By contrast, the response of mycobacteria to mitomycin C preferentially involved a RecA-dependent pathway.     

Structural basis for a novel mechanism of DNA bridging and alignment in eukaryotic DSB DNA repair

Eukaryotic DNA polymerase mu of the PolX family can promote the association of the two 3′‐protruding ends of a DNA double‐strand break (DSB) being repaired (DNA synapsis) even in the absence of the core non‐homologous end‐joining (NHEJ) machinery. Here, we show that terminal deoxynucleotidyltransferase (TdT), a closely related PolX involved in V(D)J recombination, has the same property. We solved its crystal structure with an annealed DNA synapsis containing one micro‐homology (MH) base pair and one nascent base pair. This structure reveals how the N‐terminal domain and Loop 1 of Tdt cooperate for bridging the two DNA ends, providing a templating base in trans and limiting the MH search region to only two base pairs. A network of ordered water molecules is proposed to assist the incorporation of any nucleotide independently of the in trans templating base. These data are consistent with a recent model that explains the statistics of sequences synthesized in vivo by Tdt based solely on this dinucleotide step. Site‐directed mutagenesis and functional tests suggest that this structural model is also valid for Pol mu during NHEJ.     

Factors determining DNA double-strand break repair pathway choice in G2 phase

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.     

Non-homologous end joining: Emerging themes and unanswered questions

Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation induced DNA double strand breaks in human cells. Here, we discuss current insights into the mechanism of NHEJ and the interplay between NHEJ and other pathways for repair of IR-induced DNA damage.     

Non-homologous DNA end joining in normal and cancer cells and its dependence on break structures

DNA double-strand breaks (DSBs) are a serious threat to the cell, for if not or miss-repaired, they can lead to chromosomal aberration, mutation and cancer. DSBs in human cells are repaired via non-homologous DNA end joining (NHEJ) and homologous recombination repair pathways. In the former process, the structure of DNA termini plays an important role, as does the genetic constitution of the cells, through being different in normal and pathological cells. In order to investigate the dependence of NHEJ on DSB structure in normal and cancer cells, we used linearized plasmids with various, complementary or non-complementary, single-stranded or blunt DNA termini, as well as whole-cell extract isolated from normal human lymphocytes, chronic myeloid leukemia K562 cells and lung cancer A549 cells. We observed a pronounced variability in the efficacy of NHEJ reaction depending on the type of ends. Plasmids with complementary and blunt termini were more efficiently repaired than the substrate with 3' protruding single-strand ends. The hierarchy of the effectiveness of NHEJ was on average, from the most effective to the least, A549/ normal lymphocytes/ K562. Our results suggest that the genetic constitution of the cells together with the substrate terminal structure may contribute to the efficacy of the NHEJ reaction. This should be taken into account on considering its applicability in cancer chemo- or radiotherapy by pharmacologically modulating NHEJ cellular responses.     

DNA repair pathways in human multiple myeloma

Every day, cells are faced with thousands of DNA lesions, which have to be repaired to preserve cell survival and function. DNA repair is more or less accurate and could result in genomic instability and cancer. We review here the current knowledge of the links between molecular features, treatment, and DNA repair in multiple myeloma (MM), a disease characterized by the accumulation of malignant plasma cells producing a monoclonal immunoglobulin. Genetic instability and abnormalities are two hallmarks of MM cells and aberrant DNA repair pathways are involved in disease onset, primary translocations in MM cells, and MM progression. Two major drugs currently used to treat MM, the alkylating agent Melphalan and the proteasome inhibitor Bortezomib act directly on DNA repair pathways, which are involved in response to treatment and resistance. A better knowledge of DNA repair pathways in MM could help to target them, thus improving disease treatment.     

Monoubiquitination of the nonhomologous end joining protein XRCC4

Nonhomologous end joining is one of the major pathways by which cells repair double-strand breaks, and the XRCC4-DNA ligase IV complex is required for the ligation step. To better understand the regulation and stability of XRCC4 and DNA ligase IV, we investigated the ubiquitination status of these two proteins. We identified a predominantly monoubiquitinated form of XRCC4, and higher molecular weight forms of ubiquitinated XRCC4 were detected in lower abundance. In response to etoposide-induced DNA damage, ubiquitinated XRCC4 became more pronounced and was additionally phosphorylated. We confirmed that DNA ligase IV is unstable in the absence of XRCC4, with a half-life of approximately 30-90 min. Unlike XRCC4, we did not detect ubiquitinated forms of DNA ligase IV, and we found that the presence of XRCC4 stabilized DNA ligase IV more significantly than proteasome inhibitors. Monoubiquitination of XRCC4 may play a critical role in the regulation of nonhomologous end joining.     

Nonhomologous end joining and homologous recombination DNA repair pathways in integration mutagenesis in the xylose-fermenting yeast Pichia stipitis

Pichia stipitis integrates linear homologous DNA fragments mainly ectopically. High rates of randomly occurring integration allow tagging mutagenesis with high efficiency using simply PCR amplificates of suitable selection markers from the P. stipitis genome. Linearization of an autonomously replicating vector caused a distinct increase of the transformation efficiency compared with the circular molecule. Cotransformation of a restriction endonuclease further enhanced the transformation efficiency. This effect was also observed with integrative vector DNA. In most cases vector integration in chromosomal targets did not depend on microhomologies, indicating that restriction-enzyme-mediated integration (REMI) does not play an essential role in P. stipitis. Small deletions were observed at the ends of the integrated vectors and in the target sites. Disruption of the PsKU80 gene increased the frequency of homologous integration considerably but resulted in a remarkable decrease of the transformation efficiency. These results suggest that in P. stipitis the nonhomologous end joining (NHEJ) pathway obviously predominates the homologous recombination pathway of double-strand break repair.     

Non-homologous DNA end joining in the mature rat brain

Recent evidence suggests that DNA double strand breaks (DSBs) are introduced in neurons during the course of normal development, and that repair of such DSBs is essential for neuronal survival. Here we describe a non-homologous DNA end joining (NHEJ) system in the adult rat brain that may be used to repair DNA DSBs. In the brain NHEJ system, blunt DNA ends are joined with lower efficiency than cohesive or non-matching protruding ends. Moreover, brain NHEJ is blocked by DNA ligase inhibitors or by dATP and can occur in the presence or absence of exogenously added ATP. Comparison of NHEJ activities in several tissues showed that brain and testis share similar mechanisms for DNA end joining, whereas the activity in thymus seems to utilize different mechanisms than in the nervous system. The developmental profile of brain NHEJ showed increasing levels of activity after birth, peaking at postnatal day 12 and then gradually decreasing along with age. Brain distribution analysis in adult animals showed that NHEJ activity is differentially distributed among different regions. We suggest that the DNA NHEJ system may be utilized in the postnatal brain for the repair of DNA double strand breaks introduced within the genome in the postnatal brain.     

SQSTM1/p62 mediates crosstalk between autophagy and the UPS in DNA repair

SQSTM1/p62 (sequestosome 1) selectively targets polyubiquitinated proteins for degradation via macroautophagy and the proteasome. Additionally, SQSTM1 shuttles between the cytoplasmic and nuclear compartments, although its role in the nucleus is relatively unknown. Here, we report that SQSTM1 dynamically associates with DNA damage foci (DDF) and regulates DNA repair. Upon induction of DNA damage SQSTM1 interacts with FLNA (filamin A), which has previously been shown to recruit DNA repair protein RAD51 (RAD51 recombinase) to double-strand breaks and facilitate homologous recombination (HR). SQSTM1 promotes proteasomal degradation of FLNA and RAD51 within the nucleus, resulting in reduced levels of nuclear RAD51 and slower DNA repair. SQSTM1 regulates the ratio between HR and nonhomologous end joining (NHEJ) by promoting the latter at the expense of the former. This SQSTM1-dependent mechanism mediates the effect of macroautophagy on DNA repair. Moreover, nuclear localization of SQSTM1 and its association with DDF increase with aging and are prevented by life-span-extending dietary restriction, suggesting that an imbalance in the mechanism identified here may contribute to aging and age-related diseases.     

Role of the yeast DNA repair protein Nej1 in end processing during the repair of DNA double strand breaks by non-homologous end joining

DNA double strand breaks (DSB)s often require end processing prior to joining during their repair by non-homologous end joining (NHEJ). Although the yeast proteins, Pol4, a Pol X family DNA polymerase, and Rad27, a nuclease, participate in the end processing reactions of NHEJ, the mechanisms underlying the recruitment of these factors to DSBs are not known. Here we demonstrate that Nej1, a NHEJ factor that interacts with and modulates the activity of the NHEJ DNA ligase complex (Dnl4/Lif1), physically and functionally interacts with both Pol4 and Rad27. Notably, Nej1 and Dnl4/Lif1, which also interacts with both Pol4 and Rad27, independently recruit the end processing factors to in vivo DSBs via mechanisms that are additive rather than redundant. As was observed with Dnl4/Lif1, the activities of both Pol4 and Rad27 were enhanced by the interaction with Nej1. Furthermore, Nej1 increased the joining of incompatible DNA ends in reconstituted reactions containing Pol4, Rad27 and Dnl4/Lif1, indicating that the stimulatory activities of Nej1 and Dnl4/Lif1 are also additive. Together our results reveal novel roles for Nej1 in the recruitment of Pol4 and Rad27 to in vivo DSBs and the coordination of the end processing and ligation reactions of NHEJ.     

DNA polymerase lambda can elongate on DNA substrates mimicking non-homologous end joining and interact with XRCC4-ligase IV complex

Non-homologous end joining (NHEJ) is one of two pathways responsible for the repair of double-strand breaks in eukaryotic cells. The mechanism involves the alignment of broken DNA ends with minimal homology, fill in of short gaps by DNA polymerase(s), and ligation by XRCC4-DNA ligase IV complex. The gap-filling polymerase has not yet been positively identified, but recent biochemical studies have implicated DNA polymerase lambda (pol lambda), a novel DNA polymerase that has been assigned to the pol X family, in this process. Here we demonstrate that purified pol lambda can efficiently catalyze gap-filling synthesis on DNA substrates mimicking NHEJ. By designing two truncated forms of pol lambda, we also show that the unique proline-rich region in pol lambda plays a role in limiting strand displacement synthesis, a feature that may help its participation in in vivo NHEJ. Moreover, pol lambda interacts with XRCC4-DNA ligase IV via its N-terminal BRCT domain and the interaction stimulates the DNA synthesis activity of pol lambda. Taken together, these data strongly support that pol lambda functions in DNA polymerization events during NHEJ.