Abstract
SummaryParalog 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.
Highlights
V(D)J recombination assembles immunoglobulin and T cell receptor variable exons from variable (V), diversity (D), and joining (J) gene segments via a cut-and-paste mechanism (Bassing et al, 2002)
We found that PaxxÀ/À v-abl pro-B cell clones were significantly more radiosensitive than wild-type v-abl pro-B cell lines but less sensitive than XRCC4-like factor (XLF)- and XRCC4-deficient v-abl pro-B cells (Figure 1D; Table S2)
Reminiscent of Ku80- and XRCC4-deficient pro-B cells, 20.1% of PaxxÀ/À XlfÀ/À cells contained two 53BP1 foci corresponding to DNA breaks at both Igk alleles (Lescale et al, 2016) in comparison to 3.6%, 4.3%, 6.1%, and 7.3% in Rag2À/À, WT, PaxxÀ/À, and XlfÀ/À pro-B cells, respectively (Figures 2A and 2B; Table S3). These results indicate that RAG-mediated DNA breaks are readily formed in Paralog of XRCC4 and XLF (PAXX)- and PAXX/XLF-deficient cells, and in contrast to single deficiency, repair of these DNA breaks does not seem to occur in combined PAXX/XLF deficient cells, paralleling what is seen in the absence of the canonical nonhomologous end-joining (NHEJ) factors Ku80 and XRCC4
Summary
V(D)J recombination assembles immunoglobulin and T cell receptor variable exons from variable (V), diversity (D), and joining (J) gene segments via a cut-and-paste mechanism (Bassing et al, 2002). This process occurs in developing lymphocytes during the G1 phase of the cell cycle and is initiated when the recombination-activating gene products RAG1 and RAG2 (forming the RAG endonuclease) introduce double-strand breaks (DSBs) among V, D, and J coding gene segments and flanking recombination signal sequences (RSSs) (Schatz and Swanson, 2011). The stabilization and tethering of broken DNA ends depends on ATM kinase activity and the formation of ATM-dependent DNA repair foci (Helmink and Sleckman, 2012; Kumar et al, 2014)
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