Classical Nonhomologous DNA End Joining Research Articles

Mitochondrial genome stability in human: understanding the role of DNA repair pathways.

Mitochondria are semiautonomous organelles in eukaryotic cells and possess their own genome that replicates independently. Mitochondria play a major role in oxidative phosphorylation due to which its genome is frequently exposed to oxidative stress. Factors including ionizing radiation, radiomimetic drugs and replication fork stalling can also result in different types of mutations in mitochondrial DNA (mtDNA) leading to genome fragility. Mitochondria from myopathies, dystonia, cancer patient samples show frequent mtDNA mutations such as point mutations, insertions and large-scale deletions that could account for mitochondria-associated disease pathogenesis. The mechanism by which such mutations arise following exposure to various DNA-damaging agents is not well understood. One of the well-studied repair pathways in mitochondria is base excision repair. Other repair pathways such as mismatch repair, homologous recombination and microhomology-mediated end joining have also been reported. Interestingly, nucleotide excision repair and classical nonhomologous DNA end joining are not detected in mitochondria. In this review, we summarize the potential causes of mitochondrial genome fragility, their implications as well as various DNA repair pathways that operate in mitochondria.

Homologous recombination-mediated repair of DNA double-strand breaks operates in mammalian mitochondria.

Mitochondrial DNA is frequently exposed to oxidative damage, as compared to nuclear DNA. Previously, we have shown that while microhomology-mediated end joining can account for DNA deletions in mitochondria, classical nonhomologous DNA end joining, the predominant double-strand break (DSB) repair pathway in nucleus, is undetectable. In the present study, we investigated the presence of homologous recombination (HR) in mitochondria to maintain its genomic integrity. Biochemical studies revealed that HR-mediated repair of DSBs is more efficient in the mitochondria of testes as compared to that of brain, kidney and spleen. Interestingly, a significant increase in the efficiency of HR was observed when a DSB was introduced. Analyses of the clones suggest that most of the recombinants were generated through reciprocal exchange, while~30% of recombinants were due to gene conversion in testicular extracts. Colocalization and immunoblotting studies showed the presence of RAD51 and MRN complex proteins in the mitochondria and immunodepletion of MRE11, RAD51 or NIBRIN suppressed the HR-mediated repair. Thus, our results reveal importance of homologous recombination in the maintenance of mitochondrial genome stability.

Induction of DNA damage and erroneous repair can explain genomic instability caused by endosulfan.

Endosulfan (ES) is an organochlorine pesticide, speculated to be associated with chromosomal abnormalities and diseases in humans. However, very little is known about the mechanism of its genotoxicity. Using in vivo, ex vivo and in vitro model systems, we show that exposure to ES induces reactive oxygen species (ROS) in a concentration and time-dependent manner. The generation of ROS results in DNA double-strand breaks either directly or in a replication-dependent manner, both in mice and human cells. Importantly, ES-induced DNA damage evokes DNA damage response, resulting in elevated levels of classical non-homologous DNA endjoining (NHEJ), the predominant double-strand break repair pathway in higher eukaryotes. Sequence analyses of NHEJ junctions revealed that ES treatment results in extensive processing of broken DNA, culminating in increased and long junctional deletions, thereby favoring erroneous repair. We also find that exposure to ES leads to significant increase in microhomology-mediated end joining (MMEJ), a LIGASE III-dependent alternative repair pathway. Therefore, we demonstrate that ES induces DNA damage and genomic instability, alters DNA damage response thereby promoting erroneous DNA repair.

Nuclear localization of mouse Ku70 in interphase cells and focus formation of mouse Ku70 at DNA damage sites immediately after irradiation.

To elucidate the mechanisms of DNA repair pathway is critical for developing next-generation radiotherapies and chemotherapeutic drugs for cancer. Ionizing radiation and many chemotherapeutic drugs kill tumor cells mainly by inducing DNA double-strand breaks (DSBs). The classical nonhomologous DNA-end joining (NHEJ) (C-NHEJ) pathway repairs a predominant fraction of DSBs in mammalian cells. The C-NHEJ pathway appears to start with the binding of Ku (heterodimer of Ku70 and Ku80) to DNA break ends. Therefore, recruitment of Ku to DSB sites might play a critical role in regulating NHEJ activity. Indeed, human Ku70 and Ku80 localize in the nuclei and accumulate at microirradiated DSB sites. However, the localization and regulation mechanisms of Ku70 and Ku80 homologues in animal models, such as mice and other species, have not been elucidated in detail, particularly in cells immediately after microirradiation. Here, we show that EYFP-tagged mouse Ku70 localizes in the interphase nuclei of mouse fibroblasts and epithelial cells. Furthermore, our findings indicate that EYFP-mouse Ku70 accumulates with its heterodimeric partner Ku80 immediately at laser-microirradiated DSB sites. We also confirmed that the structure of Ku70 nuclear localization signal (NLS) is highly conserved among various rodent species, such as the mouse, rat, degu and ground squirrel, supporting the idea that NLS is important for the regulation of rodent Ku70 function. Collectively, these results suggest that the mechanisms of regulating the localization and accumulation of Ku70 at DSBs might be well conserved between the mouse and human species.

Dynamic changes in subcellular localization of cattle XLF during cell cycle, and focus formation of cattle XLF at DNA damage sites immediately after irradiation.

Clinically, many chemotherapeutics and ionizing radiation (IR) have been applied for the treatment of various types of human and animal malignancies. These treatments kill tumor cells by causing DNA double-strand breaks (DSBs). Core factors of classical nonhomologous DNA-end joining (C-NHEJ) play a vital role in DSB repair. Thus, it is indispensable to clarify the mechanisms of C-NHEJ in order to develop next-generation chemotherapeutics for cancer. The XRCC4-like factor (XLF; also called Cernunnos or NHEJ1) is the lastly identified core NHEJ factor. The localization of core NHEJ factors might play a critical role in regulating NHEJ activity. The localization and function of XLF have not been elucidated in animal species other than mice and humans. Domestic cattle (Bos taurus) are the most common and vital domestic animals in many countries. Here, we show that the localization of cattle XLF changes dynamically during the cell cycle. Furthermore, EYFP-cattle XLF accumulates quickly at microirradiated sites and colocalizes with the DSB marker γH2AX. Moreover, nuclear localization and accumulation of cattle XLF at DSB sites are dependent on 12 amino acids (288–299) of the C-terminal region of XLF (XLF CTR). Furthermore, basic amino acids on the XLF CTR are highly conserved among domestic animals including cattle, goat and horses, suggesting that the CTR is essential for the function of XLF in domestic animals. These findings might be useful to develop the molecular-targeting therapeutic drug taking XLF as a target molecule for human and domestic animals.

Functional redundancy between repair factor XLF and damage response mediator 53BP1 in V(D)J recombination and DNA repair

The classical nonhomologous DNA end-joining (C-NHEJ) double-strand break (DSB) repair pathway in mammalian cells maintains genome stability and is required for V(D)J recombination and lymphocyte development. Mutations in the XLF C-NHEJ factor or ataxia telangiectasia-mutated (ATM) DSB response protein cause radiosensitivity and immunodeficiency in humans. Although potential roles for XLF in C-NHEJ are unknown, ATM activates a general DSB response by phosphorylating substrates, including histone H2AX and 53BP1, which are assembled into chromatin complexes around DSBs. In mice, C-NHEJ, V(D)J recombination, and lymphocyte development are, at most, modestly impaired in the absence of XLF or ATM, but are severely impaired in the absence of both. Redundant functions of XLF and ATM depend on ATM kinase activity; correspondingly, combined XLF and H2AX deficiency severely impairs V(D)J recombination, even though H2AX deficiency alone has little impact on this process. These and other findings suggest that XLF may provide functions that overlap more broadly with assembled DSB response factors on chromatin. As one test of this notion, we generated mice and cells with a combined deficiency for XLF and 53BP1. In this context, 53BP1 deficiency, although leading to genome instability, has only modest effects on V(D)J recombination or lymphocyte development. Strikingly, we find that combined XLF/53BP1 deficiency in mice severely impairs C-NHEJ, V(D)J recombination, and lymphocyte development while also leading to general genomic instability and growth defects. We conclude that XLF is functionally redundant with multiple members of the ATM-dependent DNA damage response in facilitating C-NHEJ and discuss implications of our findings for potential functions of these factors.

Robust chromosomal DNA repair via alternative end-joining in the absence of X-ray repair cross-complementing protein 1 (XRCC1)

Classical nonhomologous DNA end-joining (C-NHEJ), which is a major DNA double-strand break (DSB) repair pathway in mammalian cells, plays a dominant role in joining DSBs during Ig heavy chain (IgH) class switch recombination (CSR) in activated B lymphocytes. However, in B cells deficient for one or more requisite C-NHEJ factors, such as DNA ligase 4 (Lig4) or XRCC4, end-joining during CSR occurs by a distinct alternative end-joining (A-EJ) pathway. A-EJ also has been implicated in joining DSBs found in oncogenic chromosomal translocations. DNA ligase 3 (Lig3) and its cofactor XRCC1 are widely considered to be requisite A-EJ factors, based on biochemical studies or extrachromosomal substrate end-joining studies. However, potential roles for these factors in A-EJ of endogenous chromosomal DSBs have not been tested. Here, we report that Xrcc1 inactivation via conditional gene-targeted deletion in WT or XRCC4-deficient primary B cells does not have an impact on either CSR or IgH/c-myc translocations in activated B lymphocytes. Indeed, homozygous deletion of Xrcc1 does not impair A-EJ of I-SceI-induced DSBs in XRCC4-deficient pro-B-cell lines. Correspondingly, substantial depletion of Lig3 in Lig4-deficient primary B cells or B-cell lines does not impair A-EJ of CSR-mediated DSBs or formation of IgH/c-myc translocations. Our findings firmly demonstrate that XRCC1 is not a requisite factor for A-EJ of chromosomal DSBs and raise the possibility that DNA ligase 1 (Lig1) may contribute more to A-EJ than previously considered.

ATM damage response and XLF repair factor are functionally redundant in joining DNA breaks.

Classical non-homologous DNA end-joining (C-NHEJ) is a major mammalian DNA double strand break (DSB) repair pathway. Deficiencies for C-NHEJ factors, such as XRCC4, abrogate lymphocyte development, owing to a strict requirement for C-NHEJ to join V(D)J recombination DSB intermediates1,2. The XRCC4-like factor (XLF) is mutated in certain immunodeficient human patients and has been implicated in C-NHEJ3,4,5,6. Yet, XLF-deficient mice have relatively normal lymphocyte development and their lymphocytes support normal V(D)J recombination5. The Ataxia Telangiectasia-Mutated protein (“ATM”) detects DSBs and activates DSB responses by phosphorylating substrates including histone H2AX7. However, ATM-deficiency causes only modest V(D)J recombination and lymphocyte developmental defects, and H2AX-deficiency does not measurably impact these processes7,8,9. Here, we show that XLF, ATM, and H2AX all have fundamental roles in processing and joining ends during V(D)J recombination; but that these roles have been masked by unanticipated functional redundancies. Thus, combined ATM/XLF-deficiency nearly blocks mouse lymphocyte development due inability to process and join chromosomal V(D)J recombination DSB intermediates. Combined XLF and ATM deficiency also severely impairs C-NHEJ, but not alternative end-joining, during IgH class switch recombination. Redundant ATM and XLF functions in C-NHEJ are mediated via ATM kinase activity and are not required for extra-chromosomal V(D)J recombination, suggesting a role for chromatin-associated ATM substrates. Correspondingly, conditional H2AX inactivation in XLF-deficient pro-B lines leads to V(D)J recombination defects associated with marked degradation of unjoined V(D)J ends, revealing that H2AX indeed has a role in this process.

DNA-PKcs and Artemis function in the end-joining phase of immunoglobulin heavy chain class switch recombination

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Artemis are classical nonhomologous DNA end-joining (C-NHEJ) factors required for joining a subset of DNA double-strand breaks (DSB), particularly those requiring end processing. In mature B cells, activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) by introducing lesions into S regions upstream of two recombining CH exons, which are processed into DSBs and rejoined by C-NHEJ to complete CSR. The function of DNA-PKcs in CSR has been controversial with some reports but not others showing that DNA-PKcs–deficient mice are significantly impaired for CSR. Artemis-deficient B cells reportedly undergo CSR at normal levels. Overall, it is still not known whether there are any CSR-associated DSBs that require DNA-PKcs and/or Artemis to be joined. Here, we have used an immunoglobulin (Ig)H locus-specific fluorescent in situ hybridization assay to unequivocally demonstrate that both DNA-PKcs and, unexpectedly, Artemis are necessary for joining a subset of AID-dependent DSBs. In the absence of either factor, B cells activated for CSR frequently generate AID-dependent IgH locus chromosomal breaks and translocations. We also find that under specific activation conditions, DNA-PKcs−/− B cells with chromosomal breaks are eliminated or at least prevented from progressing to metaphase via a p53-dependent response.