Human Mre11/Human Rad50/Nbs1 and DNA Ligase IIIα/XRCC1 Protein Complexes Act Together in an Alternative Nonhomologous End Joining Pathway

Abstract

Recent studies have implicated a poorly defined alternative pathway of nonhomologous end joining (alt-NHEJ) in the generation of large deletions and chromosomal translocations that are frequently observed in cancer cells. Here, we describe an interaction between two factors, hMre11/hRad50/Nbs1 (MRN) and DNA ligase III␣/XRCC1, that have been linked with alt-NHEJ. Expression of DNA ligase III␣ and the association between MRN and DNA ligase III␣/XRCC1 are altered in cell lines defective in the major NHEJ pathway. Most notably, DNA damage induced the association of these factors in DNA ligase IV-deficient cells. MRN interacts with DNA ligase III␣/XRCC1, stimulating intermolecular ligation, and together these proteins join incompatible DNA ends in a reaction that mimics alt-NHEJ. Thus, our results provide novel mechanistic insights into the alt-NHEJ pathway that not only contributes to genome instability in cancer cells but may also be a therapeutic target.The repair of DNA double-strand breaks (DSBs) 3 is of the utmost importance for cell viability and genomic stability. Notably, defects in DSB repair result in genomic rearrangements that promote cancer formation and progression (1-3). Although there are multiple DSB repair pathways, these pathways can be divided into two groups depending on whether the repair reaction is dependent or not upon substantial DNA sequence homology between the recombining molecules (4, 5). The key step of the major homology-dependent pathway is the invasion of a single strand into a homologous duplex. In contrast, the key step of nonhomologous end joining (NHEJ) involves the end-to-end alignment of broken DNA molecules (4, 5).Unlike the major homology-dependent pathway, repair of DSBs by the major or classic NHEJ (C-NHEJ) pathway is errorprone, frequently resulting in the loss or addition of a few nucleotides at the break site (4, 5). Despite the mutagenic consequences of NHEJ, this is a major DSB repair pathway in mammalian cells. In addition, there is emerging evidence for alternative versions of NHEJ (alt-NHEJs) that are more errorprone than C-NHEJ. Although alt-NHEJ is more evident in cells that are deficient in C-NHEJ (6, 7), it is detectable in wild-type cells (8). Repair of DSBs by alt-NHEJ is characterized by the presence of short tracts of sequence homology (microhomologies) at the repair site, large deletions, and chromosome translocations (6 -10). Furthermore, alt-NHEJ appears to be responsible for chromosomal translocations found in cancer cells (11)(12)(13)(14)(15) and to make a much greater contribution to DSB repair in cancer cells compared with normal cells (10).A combination of biochemical and molecular genetic studies by many laboratories has identified the key components of C-NHEJ (4, 5). In contrast, the factors involved in and the mechanisms of alt-NHEJ are not well defined. Poly(ADP-ribose) polymerase-1 (PARP-1), the initiating protein for DNA single-strand break (SSB) repair, also binds to DSBs (16) and has been implicated in DNA PK-independent NHEJ (17, 18). The h...

DNA-dependent Protein Kinase Regulates DNA End Resection in Concert with Mre11-Rad50-Nbs1 (MRN) and Ataxia Telangiectasia-mutated (ATM)

“…7, A and C). This result is unexpected because MRX promotes Ku-mediated end joining in budding yeast (41), and a previous study showed that MRN strongly stimulates DNA ligation by DNA ligase III-XRCC1 by mediating DNA end tethering in vitro (50). It is possible that human MRN-mediated end synapsis promotes the activity of ligase III-XRCC1 but does not provide a configuration of ends that is favorable for the ligase IV-XRCC4 complex.…”

Chromatin modification and NBS1: their relationship in DNA double-strand break repair

“…It was hypothesized that cells deficient in XRCC4, a factor involved in classical NHEJ, would use the alt-NHEJ pathway for DSB repair; however, depletion of MRE11 markedly decreased alt-NHEJ, suggesting a role of MRE11 in this alternative pathway, most likely when it is in the form of the MRN complex (Dinkelmann et al, 2009;Rass et al, 2009). DNA ligase IIIα and XRCC1 were also reported to be involved in the alt-NHEJ pathway (Della-Maria et al, 2011). The MRN complex has been confirmed to interact with the DNA ligase IIIα/XRCC1 complex directly and to stimulate DNA ligase IIIα/XRCC1-dependent intermolecular ligation in vitro, even for incompatible DNA ends, which mirrors alt-NHEJ repair, suggesting that NBS1 and likely the MRN complex participate in the alt-NHEJ pathway.…”

X-ray repair cross-complementing protein 1 (XRCC1) deficiency enhances class switch recombination and is permissive for alternative end joining

“…XRCC1 is a DNA repair factor that has recently been implicated as a component of A-EJ (17,18). Because XRCC1 is essential for mouse embryonic development (20), there is a lack of direct genetic evidence for its involvement in CSR and A-EJ.…”

“…XRCC1 was recently implicated as a component of A-EJ (17,18). If that were the case, deletion of Xrcc1 in NHEJ-deficient cells (e.g., Lig4 Δ/Δ ) should inhibit CSR.…”

“…In addition, XRCC1 has recently been implicated in A-EJ (17,18), along with other SSB repair factors (19). To assess the role of XRCC1 in CSR and A-EJ, we deleted Xrcc1 in wild-type and Lig4-deficient CH12F3 cells by targeted mutation.…”