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

Abstract

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.T o repair DNA double-strand breaks (DSBs), mammalian cells use two major DSB repair pathways: homologous recombination (HR) and classical nonhomologous end joining (C-NHEJ). HR requires a long homologous template for repair and is active in S/G2 cell cycle phase (1). In contrast, C-NHEJ, which directly ligates DSBs with short (1-4 bp) or no homologies, functions throughout interphase, including during the G1 cell cycle phase (2-4). C-NHEJ repairs diverse DSBs, including those arising ectopically by ionizing radiation as well as DSBs generated as intermediates during V(D)J recombination in developing progenitor (pro)-B and -T lymphocytes and Ig heavy chain (IgH) class switch recombination (CSR) in activated mature B lymphocytes (5). The C-NHEJ pathway has a number of characterized members, including "core" C-NHEJ factors that were so-named in part based on their requirement for joining known types of ends via C-NHEJ and their evolutionary conservation (6). Such core factors include the Ku70 and Ku80 proteins, which form the "Ku" end recognition complex, and the XRCC4 and DNA Ligase4 proteins, which form the C-NHEJ ligation complex (5, 6).Exons that encode Ig and T-cell receptor (TCR) variable region exons are assembled from germline V, D, and J gene segments. This V(D)J recombinatio...

PAXX and Xlf interplay revealed by impaired CNS development and immunodeficiency of double KO mice

“…[8][9][10] Interestingly, asynchronous PAXXdefective CH12 B-cell lines or v-Abl-transformed Pro-B-cell lines do not demonstrate any IR sensitivity above that of their wt counterparts, as we found in MEFs, and the concomitant PAXX and Xlf deficiency does not worsen the sensitivity caused by Xlf mutation alone in these cycling cells. 13 In contrast, the synchronization of PAXX −/− /Xlf − / − v-Abl pro-B cells into the G1 phase of the cell cycle with either STI-571 (vAbl kinase inhibitor) or PD033991 (CDK4/6 inhibitor) results in an extreme sensitivity to IR, well beyond that of Xlf − / − single mutants in the same conditions arguing for a unique role of PAXX in classical NHEJ when DNA damage occurs in G1, hence in the absence of functional HR. Our observation of an IR sensitivity of small resting, ex vivo purified T lymphocytes from PAXX − / − mice concords with this conclusion of a specific role of PAXX during C-NHEJ.…”

“…[8][9][10][12][13][14][15] This suggested a possible functional complementation of PAXX deficiency in certain conditions.…”

“…11 Surprisingly, for a NHEJ factor, the deficiency of PAXX does not systematically result in an increased sensitivity to ionizing radiation (IR) and the results of the various DNA repair assays are highly controversial, depending on the experimental settings. [8][9][10][12][13][14][15] This suggested a possible functional complementation of PAXX deficiency in certain conditions.…”

Nonhomologous DNA end-joining for repair of DNA double-strand breaks

“…Both XLF and PAXX share structural similarity with XRCC4 (62,64). Individual XLF and PAXX mutants display only a mild phenotype, but XLF PAXX double mutants are synthetically lethal in mice and reduce V(D)J recombination in human B lymphocytes (98)(99)(100)(101), suggesting that while they may be redundant, at least one is necessary for efficient repair by NHEJ. The main purpose of XLF and PAXX appears to be in providing additional structural support to stabilize two DNA ends, thereby enhancing the ability of XRCC4·Lig4.…”

Synthetic lethality between PAXX and XLF in mammalian development

“…Due to the extensive cell death in lymphoid progenitors in developing embryos, we were unable to isolate B cells from Paxx −/− Xlf −/− mice to examine their radiosensitivity in vitro. However, recent data using Paxx/Xlf single-knockout and double-knockout v-Abl transformed pro-B cells derived from wild-type mice demonstrated that, in this cell lineage as in the developing CNS, a synthetic relationship between PAXX and XLF exists in terms of cellular sensitivity to IR as well as in the process of V(D)J recombination (Kumar et al 2016;Lescale et al 2016b). Thus, there appears to be a tissue-specific requirement for PAXX and XLF: In some cells, both PAXX and XLF are required for NHEJ, but, in others, either one or the other suffices to a major degree.…”