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

Abstract

The classical nonhomologous DNA end-joining (C-NHEJ) doublestrand 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.ataxia telangiectasia-mutated | double-strand DNA break repair | NHEJ1 | Cernunnos I n mammalian cells, double-strand break (DSBs) can be generated extrinsically by ionizing radiation (IR) or by general physiologic factors such as oxidative stress, DNA replication, or transcription. Programmed DSBs are introduced into antigen receptor loci during V(D)J recombination in developing lymphocytes and during Ig heavy chain (IgH) class switch recombination (CSR) in activated mature B lymphocytes. There are two major DSB repair pathways in mammalian cells (1, 2). Homologous recombination (HR) accurately repairs DSBs in the S and G2 phases of the cell cycle; classical nonhomologous DNA end-joining (C-NHEJ) fuses broken DNA ends that lack or have limited homology throughout the cell cycle but predominantly in G1. There are multiple well-characterized C-NHEJ factors (2). The Ku70/Ku80 (Ku) complex recognizes DSBs, and the XRCC4/DNA Ligase IV (Lig4) complex ligates DSBs. DNA-dependent protein kinase, catalytic subunit (DNAPKcs) is activated by Ku at DSBs and, among other functions, activates Artemis, which processes DSBs. The precise role for the XLF (also called NHEJ1 or Cernunnos) C-NHEJ factor remains unknown; however, XLF recently was found to be functionally redundant ...

Functional redundancy between the XLF and DNA-PKcs DNA repair factors in V(D)J recombination and nonhomologous DNA end joining

“…In addition, unjoined CEs and SEs generated during attempted V(D)J recombination in XLF/H2AX and XLF/53BP1 double-deficient pro-B cells are highly resected, consistent with a role for H2AX and 53BP1 in end protection (34)(35)(36). It is notable that ATM deficiency or treatment with ATM inhibitors rescues the end resection, but not the V(D)J joining phenotype of XLF/H2AX or XLF/53BP1 doubledeficient pro-B lines (31,32); this is consistent with ATM having both a role in end joining and in activating the CtIP nuclease for end resection (34). Potential roles for XLF in end protection have been speculated but not tested.…”

“…SEs and CEs are excessively resected in cells double deficient for XLF and histone H2AX, and to a lesser extent in cells deficient for XLF and ATM or XLF and 53BP1 (31,32). Although XLF apparently does not prevent increased end resection in the combined absence of ATM or H2AX and either Artemis or Lig4 (34), ability of XLF to contribute to end protection through a different mechanism has not been tested.…”

“…Single deficiency for XLF or DNA-PKcs does not lead to major defects in SE joining during chromosomal V(D)J recombination in pro-B-cell lines (16,31,32) (Fig. 2 A-C).…”

“…However, chromosomal V(D)J recombination is largely normal in developing lymphocytes and pro-B-cell lines from XLF-deficient mice (29,30) because of XLF functional redundancy with ATM and downstream substrates, including H2AX and 53BP1 in C-NHEJ (31)(32)(33). Thus, XLF/ATM and XLF/53BP1 double-deficient mice are smaller than single-deficient littermates, have a SCID phenotype associated with severely deficient V(D)J CE and SE joining, and have a greater reduction in CSR than single-deficient mice (31)(32)(33). XLF/H2AX double-deficient pro-B lines also have severely impaired chromosomal V(D)J recombination (31).…”

Programmed DNA breaks in lymphoid cells: repair mechanisms and consequences in human disease

“…This results in apoptosis, loss of TCRα locus integrity and lymphopenia 53. However, others report that the defect of 53BP1‐deficient mice in V(D)J recombination is only mild54 and to date no human mutation in this gene has been reported.…”