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

Kumar, Tadi Satish; Tellier-Lebègue, Carine; Nemoz, Clément; Drevet, Pascal; Audebert, Stéphane; Roy, Sunetra; Meek, Katheryn; Charbonnier, Jean‐Baptiste; Modesti, Mauro

doi:10.1016/j.celrep.2016.09.026

Cited by 83 publications

(89 citation statements)

References 53 publications

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“…Competitions experiments showed that the X-KBM peptide (pXLF) competes in the μM range with the XLF protein and that the A-KBM peptide (pAPLF) does not displace XLF, supporting remote sites of interactions (Figure 2e and Supplementary Figure 3c, d). Also, the C-terminus of PAXX (pPAXX) does not compete with XLF binding, in agreement with previous studies that report an interaction between PAXX C-terminus and Ku70 subunit 22,23 (Supplementary Figure 3e). …”

Section: Resultssupporting

confidence: 92%

“…Wild-type XLF onto the Ku-DNA complex showed a rapid k on (4.7 ± 1.7 10 5 M −1 s −1 ) followed by a rapid dissociation (k off = 0,09 ± 0,004s −1 ) and a corresponding Kd of 0.19 ± 0.07 μM (Figure 4a). This affinity is about 10 fold stronger than the one measured by ITC with a smaller DNA and may reflect additional interactions of XLF with DNA emerging from Ku ring as observed with PAXX 22 . LW and LE mutants showed a 2.3- and 5.1-fold weaker affinity than WT protein, respectively (Kd of 0.45 ± 0.26 μM for LW and Kd of 0.98 ± 0.15 μM for LE) Supplementary Figure 3h).…”

Section: Resultsmentioning

confidence: 68%

“…Two motifs in the C-terminus of WRN cooperate for interaction with Ku, one being A-KBM like, and the other resembling the X-KBM present on XLF (Figure 1a). In addition, we and others showed recently that PAXX (Paralog of XRCC4 and XLF) interacts with the Ku70 subunit through a third type motif that is located in its C-terminus 22,23 (Figure 1a). Despite identification of KBMs in several NHEJ factors, their respective contribution to the efficiency of DSB repair is not fully understood.…”

Section: Introductionmentioning

confidence: 65%

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XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining

Nemoz

Ropars

Frit

et al. 2018

Nat Struct Mol Biol

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111

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The Ku70-Ku80 (Ku) heterodimer binds rapidly and tightly to ends of DNA double-strand breaks and recruits several factors of the Non-Homologous End Joining (NHEJ) pathway through molecular mechanisms that remain unclear. Here, we describe the crystal structures of the Ku-binding motifs (KBM) of the NHEJ proteins APLF (A-KBM) and XLF (X-KBM) bound to a Ku-DNA complex. The two KBMs motifs bind on remote sites of Ku80 α/β domain. The X-KBM occupies an internal pocket formed after an unprecedented large outward rotation of the Ku80 α/β domain. We reveal independent recruitment at laser-irradiated sites of the APLF-interacting protein XRCC4 and of XLF through the respective binding of A- and X-KBMs to Ku80. Finally, we show that mutations on the X-KBM and A KBM binding sites in Ku80 compromises efficiency and accuracy of end-joining and cellular radiosensitivity. A- and X-KBMs may represent two initial anchorage points necessary to build the NHEJ intricate interactions network.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 68%

Section: Introductionmentioning

confidence: 65%

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XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining

Nemoz

Ropars

Frit

et al. 2018

Nat Struct Mol Biol

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111

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“…The C-terminus of PAXX (aa 199 -201) interacts with Ku, and similar to XLF mutants, PAXX mutants are more sensitive to ionizing radiation and DSB inducing agents ( Fig. 2) (64,66,67).…”

Section: The Ligase Complex Of Nhejmentioning

confidence: 99%

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

Pannunzio

Watanabe

Lieber

2018

Journal of Biological Chemistry

405

360

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Nonhomologous DNA end joining (NHEJ) is the predominant DSB repair pathway throughout the cell cycle and accounts for nearly all DSB repair outside of the S and G2 phases. NHEJ relies on Ku to thread onto DNA termini and thereby improve the affinity of the NHEJ enzymatic components consisting of polymerases (Pol m and Pol l), a nuclease (the Artemis·DNA-PKcs complex), and a ligase (XLF·XRCC4·Lig4 complex). Each of the enzymatic components is distinctive for its versatility in acting on diverse incompatible DNA end configurations coupled with a flexibility in loading order, resulting in many possible junctional outcomes from one DSB. DNA ends can either be directly ligated or, if the ends are incompatible, processed until a ligatable configuration is achieved that is often stabilized by up to 4 bp of terminal microhomology. Processing of DNA ends results in nucleotide loss or addition, explaining why DSBs repaired by NHEJ are rarely restored to their original DNA sequence. Thus, NHEJ is a single pathway with multiple enzymes at its disposal to repair DSBs, resulting in a diversity of repair outcomes.

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“…PAXX is recruited to DNAdsb and is a physical interactor of the Ku/DNAPK complex, notably through its interaction with Ku70. 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.…”

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confidence: 99%

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

et al. 2017

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The repair of DNA double-stranded breaks (DNAdsb) through non-homologous end joining (NHEJ) is a prerequisite for the proper development of the central nervous system and the adaptive immune system. Yet, mice with Xlf or PAXX loss of function are viable and present with very mild immune phenotypes, although their lymphoid cells are sensitive to ionizing radiation attesting for the role of these factors in NHEJ. In contrast, we show here that mice defective for both Xlf and PAXX are embryonically lethal owing to a massive apoptosis of post-mitotic neurons, a situation reminiscent to XRCC4 or DNA Ligase IV KO conditions. The development of the adaptive immune system in Xlf − / − PAXX −/− E18.5 embryos is severely affected with the block of B-and T-cell maturation at the stage of IgH and TCRβ gene rearrangements, respectively. This damaging phenotype highlights the functional nexus between Xlf and PAXX, which is critical for the completion of NHEJ-dependent mechanisms during mouse development. Cell Death and Differentiation (2018) 25, 444-452; doi:10.1038/cdd.2017; published online 27 October 2017All living organisms are subjected to multitude sources of DNA damage during their lifespan, either as a result of external assault or endogenous physiological processes.1 Among endogenous sources of physiological DNAdsb is the somatic rearrangement of immunoglobulin (Ig) and TCR genes in B and T lymphocytes, respectively, during the diversification of the adaptive immune system through V(D)J recombination.2 DNA double-stranded breaks (DNAdsb) are considered the most toxic lesions. DNAdsbs are repaired by two main mechanisms: the homologous recombination (HR) in cycling cells, when a sister chromatid is available as DNA repair template, and the non-homologous end joining (NHEJ) during all phases of the cell cycle.NHEJ proceeds via the simple religation of DNA ends without the need for a repair template.3 Briefly, the NHEJ is composed of seven core factors comprising the Ku70/80/ DNA-PKcs (DNA-dependent protein kinase catalytic subunit) complex, which recognizes and protects the broken DNA ends, the Artemis endo/exonuclease, which participates, when needed, in processing the DNA ends and the XRCC4/ DNA-Ligase IV/Xlf complex, which ultimately reseals the DNA break. The critical function of the NHEJ apparatus in various aspects of higher eukaryote development has been extensively perceived in several animal and human pathological conditions. As emblematic examples, loss of function of either XRCC4 or DNA ligase IV results in embryonic lethality in mice 4,5 and mutations in Artemis or DNA-PKcs result in severe combined immunodeficiency conditions in both men and mice, owing to aborted V(D)J recombination. 6 In addition, defects in NHEJ results in genetic instability and the propensity to develop various types of cancers, notably leukemia and lymphomas. Recently, a new DNA repair factor, PAXX (PAralog of XRCC4 and Xlf, also known as C9orf142 or XLS), has been identified independently by three laboratories based on bioinformatic...

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

Cited by 83 publications

References 53 publications

XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining

XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining

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

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

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