Absence of XRCC4 and its paralogs in human cells reveal differences in outcomes for DNA repair and V(D)J recombination

Brian Ruis, Amy Molan, Taylor Takasugi, Eric A. Hendrickson

Research output: Contribution to journalArticle

Abstract

The repair of DNA double-stranded breaks (DSBs) is an essential function performed by the Classical Non-Homologous End-Joining (C-NHEJ) pathway in higher eukaryotes. C-NHEJ, in fact, does double duty as it is also required for the repair of the intermediates formed during lymphoid B- and T-cell recombination. Consequently, the failure to properly repair DSBs leads to both genomic instability and immunodeficiency. A critical DSB protein required for C-NHEJ is the DNA Ligase IV (LIGIV) accessory factor, X-Ray Cross Complementing 4 (XRCC4). XRCC4 is believed to stabilize LIGIV, participate in LIGIV activation, and to help tether the broken DSB ends together. XRCC4′s role in these processes has been muddied by the identification of two additional XRCC4 paralogs, XRCC4-Like Factor (XLF), and Paralog of XRCC4 and XLF (PAXX). The roles that these paralogs play in C-NHEJ is partially understood, but, in turn, has itself been obscured by species-specific differences observed in the absence of one or the other paralogs. In order to investigate the role(s) that XRCC4 may play, with or without XLF and/or PAXX, in lymphoid variable(diversity)joining [V(D)J] recombination as well as in DNA DSB repair in human somatic cells, we utilized gene targeting to inactivate the XRCC4 gene in both parental and XLF HCT116 cells and then inactivated PAXX in those same cell lines. The loss of XRCC4 expression by itself led, as anticipated, to increased sensitivity to DNA damaging agents as well as an increased dependence on microhomology-mediated DNA repair whether in the context of DSB repair or during V(D)J recombination. The additional loss of XLF in these cell lines sensitized the cells even more whereas the presence or absence of PAXX was scarcely negligible. These studies demonstrate that, of the three LIG4 accessory factor paralogs, the absence of XRCC4 influences DNA repair and recombination the most in human cells.

Original languageEnglish (US)
Article number102738
JournalDNA Repair
Volume85
DOIs
StatePublished - Jan 2020

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Joining
DNA Repair
Genetic Recombination
Repair
Cells
X-Rays
X rays
DNA
Double-Stranded DNA Breaks
Accessories
Ligases
Genes
DNA Ligases
HCT116 Cells
Cell Line
Factor X
T-cells
Gene Targeting
Genomic Instability
Eukaryota

Keywords

  • Alternative-EJ (aEJ)
  • Classic non-homologous end joining (C-NHEJ)
  • Paralog of XRCC4 and XLF (PAXX)
  • Variable(Diversity)Joining recombination [V(D)J recombination]
  • X-ray cross complementing 4 (XRCC4)
  • XRCC4-like factor (XLF)

Cite this

Absence of XRCC4 and its paralogs in human cells reveal differences in outcomes for DNA repair and V(D)J recombination. / Ruis, Brian; Molan, Amy; Takasugi, Taylor; Hendrickson, Eric A.

In: DNA Repair, Vol. 85, 102738, 01.2020.

Research output: Contribution to journalArticle

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abstract = "The repair of DNA double-stranded breaks (DSBs) is an essential function performed by the Classical Non-Homologous End-Joining (C-NHEJ) pathway in higher eukaryotes. C-NHEJ, in fact, does double duty as it is also required for the repair of the intermediates formed during lymphoid B- and T-cell recombination. Consequently, the failure to properly repair DSBs leads to both genomic instability and immunodeficiency. A critical DSB protein required for C-NHEJ is the DNA Ligase IV (LIGIV) accessory factor, X-Ray Cross Complementing 4 (XRCC4). XRCC4 is believed to stabilize LIGIV, participate in LIGIV activation, and to help tether the broken DSB ends together. XRCC4′s role in these processes has been muddied by the identification of two additional XRCC4 paralogs, XRCC4-Like Factor (XLF), and Paralog of XRCC4 and XLF (PAXX). The roles that these paralogs play in C-NHEJ is partially understood, but, in turn, has itself been obscured by species-specific differences observed in the absence of one or the other paralogs. In order to investigate the role(s) that XRCC4 may play, with or without XLF and/or PAXX, in lymphoid variable(diversity)joining [V(D)J] recombination as well as in DNA DSB repair in human somatic cells, we utilized gene targeting to inactivate the XRCC4 gene in both parental and XLF− HCT116 cells and then inactivated PAXX in those same cell lines. The loss of XRCC4 expression by itself led, as anticipated, to increased sensitivity to DNA damaging agents as well as an increased dependence on microhomology-mediated DNA repair whether in the context of DSB repair or during V(D)J recombination. The additional loss of XLF in these cell lines sensitized the cells even more whereas the presence or absence of PAXX was scarcely negligible. These studies demonstrate that, of the three LIG4 accessory factor paralogs, the absence of XRCC4 influences DNA repair and recombination the most in human cells.",
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AB - The repair of DNA double-stranded breaks (DSBs) is an essential function performed by the Classical Non-Homologous End-Joining (C-NHEJ) pathway in higher eukaryotes. C-NHEJ, in fact, does double duty as it is also required for the repair of the intermediates formed during lymphoid B- and T-cell recombination. Consequently, the failure to properly repair DSBs leads to both genomic instability and immunodeficiency. A critical DSB protein required for C-NHEJ is the DNA Ligase IV (LIGIV) accessory factor, X-Ray Cross Complementing 4 (XRCC4). XRCC4 is believed to stabilize LIGIV, participate in LIGIV activation, and to help tether the broken DSB ends together. XRCC4′s role in these processes has been muddied by the identification of two additional XRCC4 paralogs, XRCC4-Like Factor (XLF), and Paralog of XRCC4 and XLF (PAXX). The roles that these paralogs play in C-NHEJ is partially understood, but, in turn, has itself been obscured by species-specific differences observed in the absence of one or the other paralogs. In order to investigate the role(s) that XRCC4 may play, with or without XLF and/or PAXX, in lymphoid variable(diversity)joining [V(D)J] recombination as well as in DNA DSB repair in human somatic cells, we utilized gene targeting to inactivate the XRCC4 gene in both parental and XLF− HCT116 cells and then inactivated PAXX in those same cell lines. The loss of XRCC4 expression by itself led, as anticipated, to increased sensitivity to DNA damaging agents as well as an increased dependence on microhomology-mediated DNA repair whether in the context of DSB repair or during V(D)J recombination. The additional loss of XLF in these cell lines sensitized the cells even more whereas the presence or absence of PAXX was scarcely negligible. These studies demonstrate that, of the three LIG4 accessory factor paralogs, the absence of XRCC4 influences DNA repair and recombination the most in human cells.

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KW - X-ray cross complementing 4 (XRCC4)

KW - XRCC4-like factor (XLF)

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