Plant PAXX has an XLF-like function and stimulates DNA end joining by the Ku-DNA ligase IV/XRCC4 complex.

Non-homologous end joining (NHEJ) plays a major role in repairing DNA double-strand breaks and is key to genome stability and editing. The minimal core NHEJ proteins, namely Ku70, Ku80, DNA ligase IV and XRCC4, are conserved, but other factors vary in different eukaryote groups. In plants, the only known NHEJ proteins are the core factors, while the molecular mechanism of plant NHEJ remains unclear. Here, we report a previously unidentified plant ortholog of PAXX, the crystal structure of which showed a similar fold to human 'PAXX'. However, plant PAXX has similar molecular functions to human XLF, by directly interacting with Ku70/80 and XRCC4. This suggests that plant PAXX combines the roles of mammalian PAXX and XLF and that these functions merged into a single protein during evolution. This is consistent with a redundant function of PAXX and XLF in mammals.

Synthetic lethality between PAXX and XLF in mammalian development.

PAXX was identified recently as a novel nonhomologous end-joining DNA repair factor in human cells. To characterize its physiological roles, we generated Paxx-deficient mice. Like Xlf-/- mice, Paxx-/- mice are viable, grow normally, and are fertile but show mild radiosensitivity. Strikingly, while Paxx loss is epistatic with Ku80, Lig4, and Atm deficiency, Paxx/Xlf double-knockout mice display embryonic lethality associated with genomic instability, cell death in the central nervous system, and an almost complete block in lymphogenesis, phenotypes that closely resemble those of Xrcc4-/- and Lig4-/- mice. Thus, combined loss of Paxx and Xlf is synthetic-lethal in mammals.

PAXX is a novel target to overcome resistance to doxorubicin and cisplatin in osteosarcoma.

Osteosarcoma (OS) is the most common primary malignant bone tumor diagnosed in children and adolescents. Unfortunately, OS patients with metastatic or recurrent disease are highly resistant to front line chemotherapy that significantly limits the long-term survival rate. Since majority of chemotherapeutic agents used in OS work by generating DNA damages, enhanced DNA repair pathways are generally associated with chemoresistance in OS treatment. However, the exact mechanisms of chemoresistance in OS are not fully understood. Our study found that paralogue of XRCC4 and XLF (PAXX), which was identified recently as a novel factor of non-homologous end joining (NHEJ), is upregulated in chemoresistant OS cells. Importantly, PAXX deficiency re-sensitizes chemoresistant OS cells to doxorubicin and cisplatin. Mechanistically, chemoresistance to doxorubicin or cisplatin results in enhanced PAXX-Ku70 interaction and elevated NHEJ efficiency. We also identified a small molecule M11 that interrupts PAXX-Ku70 interaction and re-sensitizes chemoresistant OS cells to doxorubicin and cisplatin. Thus, our data provide evidence that PAXX could serve as a novel target to overcome chemoresistance in OS treatment.

PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line.

Classical nonhomologous end joining (C-NHEJ) is a major mammalian DNA double-strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation. The XRCC4-like-factor (XLF) C-NHEJ protein is dispensable for V(D)J recombination in normal cells, but because of functional redundancy, it is absolutely required for this process in cells deficient for the ataxia telangiectasia-mutated (ATM) DSB response factor. The recently identified paralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to ionizing radiation-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B-cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B-cell line, and dispensable for normal ionizing radiation resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to ionizing radiation exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested pro-B-cell lines. Finally, we also find that PAXX deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ.

Non-homologous end joining: advances and frontiers.

DNA double-strand breaks (DSBs) are the most serious form of DNA damage. In human cells, non-homologous end joining (NHEJ) is the major pathway for the repair of DSBs. Different types of DSBs result in different subsets of NHEJ repair strategies. These variations in NHEJ repair strategies depend on numerous elements, such as the flexible recruitment of NHEJ-related proteins, the complexity of the DSB ends, and the spatial- and temporal-ordered formation of the multi-protein complex. On the one hand, current studies of DNA DSBs repair focus on the repair pathway choices between homologous recombination and classic or alternative NHEJ. On the other hand, increasing researches have also deepened the significance and dug into the cross-links between the NHEJ pathway and the area of genome organization and aging. Although remarkable progress has been made in elucidating the underlying principles during the past decades, the detailed mechanism of action in response to different types of DSBs remains largely unknown and needs further evaluation in the future study.

Helicobacter pylori plus N-Methyl-N'-nitro-N-nitrosoguanidine: DNA damage and repair: Malignant transformation of human esophageal epithelial cells.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG), found in pickled foods and in chlorinated water, has been used to induce malignant transformation and gastrointestinal cancer in rats. Helicobacter pylori (HP) is implicated in human gastric cancer and possibly also in esophageal cancer. These two agents - one chemical and the other biological - might act together to induce esophageal cancer. In this study, human esophageal epithelial cells (HEECs) were divided into four groups: HP, MNNG, HP + MNNG, and control. The HP-to-HEEC ratio was 100:1. Cells were exposed for 6 h and then passaged until malignant transformation. HEEC at early, intermediate, and late stages of malignant transformation were used for proliferation, cell-cycle, and invasion assays. The alkaline comet assay was performed and expression of proteins, including γ-H2AX and PAXX, was studied by western blotting, to explore DNA damage and repair processes. Measurements of cell morphology, soft-agar clone formation, and invasiveness, and a nude mouse xenograft model, were used to examine malignancy. The effect of HP was stronger than that of MNNG. The combination HP + MNNG exerted a stronger malignant transformation effect than either HP or MNNG alone. Mechanisms of this combined carcinogenesis may include promotion of cell proliferation, perturbation of the cell cycle, promotion of invasiveness, DNA double-strand break induction, or PAXX inhibition.

Role of Paralogue of XRCC4 and XLF in DNA Damage Repair and Cancer Development.

Non-homologous end joining (cNHEJ) is a major pathway to repair double-strand breaks (DSBs) in DNA. Several core cNHEJ are involved in the progress of the repair such as KU70 and 80, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Artemis, X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, and XRCC4-like factor (XLF). Recent studies have added a number of new proteins during cNHEJ. One of the newly identified proteins is Paralogue of XRCC4 and XLF (PAXX), which acts as a scaffold that is required to stabilize the KU70/80 heterodimer at DSBs sites and promotes the assembly and/or stability of the cNHEJ machinery. PAXX plays an essential role in lymphocyte development in XLF-deficient background, while XLF/PAXX double-deficient mouse embryo died before birth. Emerging evidence also shows a connection between the expression levels of PAXX and cancer development in human patients, indicating a prognosis role of the protein. This review will summarize and discuss the function of PAXX in DSBs repair and its potential role in cancer development.

PAXX, Not NHEJ1 Is an Independent Prognosticator in Colon Cancer.

Classical Non-homologous End Joining (NHEJ) pathway is the mainstay of cellular response to DNA double strand breaks. While aberrant expression of genes involved in this pathway has been linked with genomic instability and drug resistance in several cancers, limited information is available about its clinical significance in colon cancer. We performed a comprehensive analysis of seven essential genes, including XRCC5, XRCC6, PRKDC, LIG4, XRCC4, NHEJ1, and PAXX of this pathway, in colon cancer using multi-omics datasets, and studied their associations with molecular and clinicopathological features, including age, gender, stage, KRAS mutation, BRAF mutation, microsatellite instability status and promoter DNA methylation in TCGA colon cancer dataset. This analysis revealed upregulation of XRCC5, PRKDC, and PAXX in colon cancer compared to normal colon tissues, while LIG4 and NHEJ1 (XLF) displayed downregulation. The expression of these genes was independent of age and KRAS status, while XRCC5, PRKDC, and LIG4 exhibited reduced expression in BRAF mutant tumors. Interestingly, we observed a strong association between XRCC6, XRCC5, PRKDC and LIG4 overexpression and microsatellite instability status of the tumors. In multivariate analysis, high PAXX expression emerged as an independent prognostic marker for poor overall and disease specific survival. We also observed hypomethylation of PAXX promoter in tumors, which exhibited a strong correlation with its overexpression. Furthermore, PAXX overexpression was also associated with several oncogenic pathways as well as a reduction in numbers of tumor-infiltrating lymphocytes.

Absence of XRCC4 and its paralogs in human cells reveal differences in outcomes for DNA repair and V(D)J recombination.

The repair of DNA double-stranded breaks (DSBs) is an essential function performed by the Classical Non-Homologous End-Joining (C-NHEJ) pathway in higher eukaryotes. C-NHEJ, in fact, does double duty as it is also required for the repair of the intermediates formed during lymphoid B- and T-cell recombination. Consequently, the failure to properly repair DSBs leads to both genomic instability and immunodeficiency. A critical DSB protein required for C-NHEJ is the DNA Ligase IV (LIGIV) accessory factor, X-Ray Cross Complementing 4 (XRCC4). XRCC4 is believed to stabilize LIGIV, participate in LIGIV activation, and to help tether the broken DSB ends together. XRCC4's role in these processes has been muddied by the identification of two additional XRCC4 paralogs, XRCC4-Like Factor (XLF), and Paralog of XRCC4 and XLF (PAXX). The roles that these paralogs play in C-NHEJ is partially understood, but, in turn, has itself been obscured by species-specific differences observed in the absence of one or the other paralogs. In order to investigate the role(s) that XRCC4 may play, with or without XLF and/or PAXX, in lymphoid variable(diversity)joining [V(D)J] recombination as well as in DNA DSB repair in human somatic cells, we utilized gene targeting to inactivate the XRCC4 gene in both parental and XLF- HCT116 cells and then inactivated PAXX in those same cell lines. The loss of XRCC4 expression by itself led, as anticipated, to increased sensitivity to DNA damaging agents as well as an increased dependence on microhomology-mediated DNA repair whether in the context of DSB repair or during V(D)J recombination. The additional loss of XLF in these cell lines sensitized the cells even more whereas the presence or absence of PAXX was scarcely negligible. These studies demonstrate that, of the three LIG4 accessory factor paralogs, the absence of XRCC4 influences DNA repair and recombination the most in human cells.

PAXX Participates in Base Excision Repair via Interacting with Pol ß and Contributes to TMZ Resistance in Glioma Cells.

Non-homologous end joining (NHEJ) is one of the major DNA repair pathway in mammalian cell that can ligate a variety of DNA ends. However, how does all NHEJ factors communicate and organize together to achieve the final repair is still not clear. PAralog of XRCC4 and XLF (PAXX) was a new factor identified recently that play an important role in NHEJ. PAXX contributes to efficient NHEJ by interacting with Ku, which is a NHEJ key factor, and PAXX deficiency cause sensitivity to DNA double-strand break repair (DSBR). We observed that PAXX-deficient cells showed slight increase of homologous recombination (HR, which is another major DSBR repair pathways in mammalian cells). More importantly, we found that PAXX contributes to base excision repair pathway via interaction of polymerase beta (pol ß). Temozolomide (TMZ) is one of the standard chemotherapies widely applied in glioblastoma. However, TMZ resistance and lack of potent chemotherapy agents can substitute TMZ. We observed that PAXX deficiency cause more sensitivity to TMZ-resistant glioma cells. In conclusion, the PAXX contributes to a variety of DNA repair pathways and TMZ resistance. Therefore, inhibition of PAXX may provide a promising way to overcome TMZ resistance and improve TMZ therapeutic effects in glioma treatment.

The Non-Homologous End Joining Protein PAXX Acts to Restrict HSV-1 Infection.

Herpes simplex virus 1 (HSV-1) has extensive interactions with the host DNA damage response (DDR) machinery that can be either detrimental or beneficial to the virus. Proteins in the homologous recombination pathway are known to be required for efficient replication of the viral genome, while different members of the classical non-homologous end-joining (c-NHEJ) pathway have opposing effects on HSV-1 infection. Here, we have investigated the role of the recently-discovered c-NHEJ component, PAXX (Paralogue of XRCC4 and XLF), which we found to be excluded from the nucleus during HSV-1 infection. We have established that cells lacking PAXX have an intact innate immune response to HSV-1 but show a defect in viral genome replication efficiency. Counterintuitively, PAXX-/- cells were able to produce greater numbers of infectious virions, indicating that PAXX acts to restrict HSV-1 infection in a manner that is different from other c-NHEJ factors.

Deficiency of XLF and PAXX prevents DNA double-strand break repair by non-homologous end joining in lymphocytes.

Non-homologous end joining (NHEJ) is a major DNA double-strand break (DSB) repair pathway that functions in all phases of the cell cycle. NHEJ repairs genotoxic and physiological DSBs, such as those generated by ionizing radiation and during V(D)J recombination at antigen receptor loci, respectively. DNA end joining by NHEJ relies on the core factors Ku70, Ku80, XRCC4, and DNA Ligase IV. Additional proteins also play important roles in NHEJ. The XRCC4-like factor (XLF) participates in NHEJ through its interaction with XRCC4, and XLF deficiency in humans leads to immunodeficiency and increased sensitivity to ionizing radiation. However, XLF is dispensable for NHEJ-mediated DSB repair during V(D)J recombination in murine lymphocytes, where it may have redundant functions with other DSB repair factors. Paralog of XRCC4 and XLF (PAXX) is a recently identified NHEJ factor that has structural similarity to XRCC4 and XLF. Here we show that PAXX is also dispensable for NHEJ during V(D)J recombination and during the repair of genotoxic DSBs in lymphocytes. However, a combined deficiency of PAXX and XLF blocks NHEJ with a severity comparable to that observed in DNA Ligase IV-deficient cells. Similar to XLF, PAXX interacts with Ku through its C-terminal region, and mutations that disrupt Ku binding prevent PAXX from promoting NHEJ in XLF-deficient lymphocytes. Our findings suggest that the PAXX and XLF proteins may have redundant functions during NHEJ.

Specific Roles of XRCC4 Paralogs PAXX and XLF during V(D)J Recombination.

Paralog of XRCC4 and XLF (PAXX) is a member of the XRCC4 superfamily and plays a role in nonhomologous end-joining (NHEJ), a DNA repair pathway critical for lymphocyte antigen receptor gene assembly. Here, we find that the functions of PAXX and XLF in V(D)J recombination are masked by redundant joining activities. Thus, combined PAXX and XLF deficiency leads to an inability to join RAG-cleaved DNA ends. Additionally, we demonstrate that PAXX function in V(D)J recombination depends on its interaction with Ku. Importantly, we show that, unlike XLF, the role of PAXX during the repair of DNA breaks does not overlap with ATM and the RAG complex. Our findings illuminate the role of PAXX in V(D)J recombination and support a model in which PAXX and XLF function during NHEJ repair of DNA breaks, whereas XLF, the RAG complex, and the ATM-dependent DNA damage response promote end joining by stabilizing DNA ends.

PAXX Is an Accessory c-NHEJ Factor that Associates with Ku70 and Has Overlapping Functions with XLF.

In mammalian cells, classical non-homologous end joining (c-NHEJ) is critical for DNA double-strand break repair induced by ionizing radiation and during V(D)J recombination in developing B and T lymphocytes. Recently, PAXX was identified as a c-NHEJ core component. We report here that PAXX-deficient cells exhibit a cellular phenotype uncharacteristic of a deficiency in c-NHEJ core components. PAXX-deficient cells display normal sensitivity to radiomimetic drugs, are proficient in transient V(D)J recombination assays, and do not shift toward higher micro-homology usage in plasmid repair assays. Although PAXX-deficient cells lack c-NHEJ phenotypes, PAXX forms a stable ternary complex with Ku bound to DNA. Formation of this complex involves an interaction with Ku70 and requires a bare DNA extension for stability. Moreover, the relatively weak Ku-dependent stimulation of LIG4/XRCC4 activity by PAXX is unmasked by XLF ablation. Thus, PAXX plays an accessory role during c-NHEJ that is largely overlapped by XLF's function.