Identification of DNA-PKcs phosphorylation sites in XRCC4 and effects of mutations at these sites on DNA end joining in a cell-free system
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Non-homologous end joining
DNA-PKcs
Serine hydroxymethyltransferase
Non-homologous end joining
Ku80
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DNA double-strand breaks (DSBs) are highly cytotoxic lesions, and unrepaired or misrepaired DSBs can lead to various human diseases, including immunodeficiency, neurological abnormalities, growth retardation, and cancer. Nonhomologous end joining (NHEJ) is the major DSB repair pathway in mammals. Ku70 and Ku80 are DSB sensors that facilitate the recruitment of downstream factors, including protein kinase DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), structural components [X-ray repair cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and paralogue of XRCC4 and XLF (PAXX)], and DNA ligase IV (LIG4), which complete DNA repair. DSBs also trigger the activation of the DNA damage response pathway, in which protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates multiple substrates, including histone H2AX. Traditionally, research on NHEJ factors was performed using in vivo mouse models and murine cells. However, the current knowledge of the genetic interactions between NHEJ factors in human cells is incomplete. Here, we obtained genetically modified human HAP1 cell lines, which lacked one or two NHEJ factors, including LIG4, XRCC4, XLF, PAXX, DNA-PKcs, DNA-PKcs/XRCC4, and DNA-PKcs/PAXX. We examined the genomic instability of HAP1 cells, as well as their sensitivity to DSB-inducing agents. In addition, we determined the genetic interaction between XRCC4 paralogues (XRCC4, XLF, and PAXX) and DNA-PKcs. We found that in human cells, XLF, but not PAXX or XRCC4, genetically interacts with DNA-PKcs. Moreover, ATM possesses overlapping functions with DNA-PKcs, XLF, and XRCC4, but not with PAXX in response to DSBs. Finally, NHEJ-deficient HAP1 cells show increased chromosomal and chromatid breaks, when compared to the WT parental control. Overall, we found that HAP1 is a suitable model to study the genetic interactions in human cells.
Ku80
Non-homologous end joining
DNA-PKcs
Ku70
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Non-homologous end joining
Ku80
DDB1
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Non-homologous end joining
Ku80
DNA polymerase mu
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In the nonhomologous end joining pathway of DNA double-strand break repair, the ligation step is catalyzed by a complex of XRCC4 and DNA ligase IV. Extracts of CHO-K1 cells are able to accurately rejoin a site-specific free radical-mediated double-strand break with partially complementary overhangs, by a mechanism involving alignment-based gap filling followed by ligation. Extracts of XR-1 cells, which lack XRCC4 and DNA ligase IV, carried out neither gap filling nor ligation. Supplementation of the extracts with recombinant XRCC4/ligase IV, but not with XRCC4 alone, restored gap filling and accurate end joining. The results imply that XRCC4 and ligase IV are essential for alignment-based gap filling, as well as for final ligation of the breaks.
Non-homologous end joining
DNA Ligases
DNA polymerase mu
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Non-homologous end joining
Ku80
XRCC1
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Ku80
Ku70
DNA-PKcs
Non-homologous end joining
DNA polymerase mu
DDB1
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Nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks created by ionizing radiation or V(D)J recombination of the immunoglobulin genes. The breaks often leave mismatched or nonligatable ends, and NHEJ must repair the breaks with high efficiency and minimal nucleotide loss. Here, the NHEJ proteins Ku, DNA-dependent protein kinase catalytic subunit, XRCC4/Ligase IV, and Cernunnos/XRCC4-like factor joined mismatched and noncohesive DNA ends in the absence of processing factors. Depending on the mismatch, Cernunnos stimulated joining 8- to 150-fold. For substrates with a blunt end and a 3′ overhanging end, Ku, XRCC4/Ligase IV, and Cernunnos ligated the 3′ overhanging hydroxyl group to the 5′ phosphate of the blunt end, leaving the other strand unjoined. This activity provides a mechanism for retaining 3′ overhang sequences, as observed during V(D)J recombination in vivo . Thus, Cernunnos/XRCC4-like factor promotes a mismatched end (MEnd) DNA ligase activity to facilitate joining and to preserve DNA sequence. Furthermore, MEnd ligase activity may have applications in recombinant DNA technology.
Non-homologous end joining
V(D)J recombination
Ku80
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Ku70
Ku80
Non-homologous end joining
DNA-PKcs
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XRCC4 and DNA Ligase 4 (LIG4) form a tight complex that provides DNA ligase activity for classical non-homologous end joining (the predominant DNA double-strand break repair pathway in higher eukaryotes) and is stimulated by XLF. Independently of LIG4, XLF also associates with XRCC4 to form filaments that bridge DNA. These XRCC4/XLF complexes rapidly load and connect broken DNA, thereby stimulating intermolecular ligation. XRCC4 and XLF both include disordered C-terminal tails that are functionally dispensable in isolation but are phosphorylated in response to DNA damage by DNA-PK and/or ATM. Here we concomitantly modify the tails of XRCC4 and XLF by substituting fourteen previously identified phosphorylation sites with either alanine or aspartate residues. These phospho-blocking and -mimicking mutations impact both the stability and DNA bridging capacity of XRCC4/XLF complexes, but without affecting their ability to stimulate LIG4 activity. Implicit in this finding is that phosphorylation may regulate DNA bridging by XRCC4/XLF filaments.
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