A XRCC4 mutant mouse, a model for human X4 syndrome, reveals interplays with Xlf, PAXX, and ATM in lymphoid development

We developed a Xrcc4M61R separation of function mouse line to overcome the embryonic lethality of Xrcc4 deficient mice. XRCC4M61R protein does not interact with Xlf, thus obliterating XRCC4-Xlf filament formation while preserving the ability to stabilize DNA Ligase IV. X4M61R mice, which are DNA repair deficient, phenocopy the Nhej1-/- (known as Xlf -/-) setting with a minor impact on the development of the adaptive immune system. The core NHEJ DNA repair factor XRCC4 is therefore not mandatory for V(D)J recombination aside from its role in stabilizing DNA ligase IV. In contrast, Xrcc4M61R mice crossed on Paxx-/-, Nhej1-/-, or Atm-/- backgrounds are severely immunocompromised, owing to aborted V(D)J recombination as in Xlf-Paxx and Xlf-Atm double KO settings. Furthermore, massive apoptosis of post-mitotic neurons causes embryonic lethality of Xrcc4M61R -Nhej1-/- double mutants. These in vivo results reveal new functional interplays between XRCC4 and PAXX, ATM and Xlf in mouse development and provide new insights in the understanding of the clinical manifestations of human XRCC4 deficient condition, in particular its absence of immune deficiency.

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Late embryonic lethality and impaired V (D)J recombination in mice lacking DNA ligase IV

Nature ◽

10.1038/24172 ◽

1998 ◽

Vol 396 (6707) ◽

pp. 173-177 ◽

Cited By ~ 394

Author(s):

Karen M. Frank ◽

JoAnn M. Sekiguchi ◽

Katherine J. Seidl ◽

Wojciech Swat ◽

Gary A. Rathbun ◽

...

Keyword(s):

Embryonic Lethality ◽

Dna Ligase ◽

Dna Ligase Iv ◽

Ligase Iv

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Molecular cloning and expression of human cDNAs encoding a novel DNA ligase IV and DNA ligase III, an enzyme active in DNA repair and recombination.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3206 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3206-3216 ◽

Cited By ~ 164

Author(s):

Y F Wei ◽

P Robins ◽

K Carter ◽

K Caldecott ◽

D J Pappin ◽

...

Keyword(s):

Dna Repair ◽

Cdna Clone ◽

Dna Ligase ◽

Dna Ligases ◽

Dna Ligase Iv ◽

Ligase Iv ◽

Dna Ligase I ◽

Cloned Cdna ◽

Dna Ligase Iii

Three distinct DNA ligases, I to III, have been found previously in mammalian cells, but a cloned cDNA has been identified only for DNA ligase I, an essential enzyme active in DNA replication. A short peptide sequence conserved close to the C terminus of all known eukaryotic DNA ligases was used to search for additional homologous sequences in human cDNA libraries. Two different incomplete cDNA clones that showed partial homology to the conserved peptide were identified. Full-length cDNAs were obtained and expressed by in vitro transcription and translation. The 103-kDa product of one cDNA clone formed a characteristic complex with the XRCC1 DNA repair protein and was identical with the previously described DNA ligase III. DNA ligase III appears closely related to the smaller DNA ligase II. The 96-kDa in vitro translation product of the second cDNA clone was also shown to be an ATP-dependent DNA ligase. A fourth DNA ligase (DNA ligase IV) has been purified from human cells and shown to be identical to the 96-kDa DNA ligase by unique agreement between mass spectrometry data on tryptic peptides from the purified enzyme and the predicted open reading frame of the cloned cDNA. The amino acid sequences of DNA ligases III and IV share a related active-site motif and several short regions of homology with DNA ligase I, other DNA ligases, and RNA capping enzymes. DNA ligases III and IV are encoded by distinct genes located on human chromosomes 17q11.2-12 and 13q33-34, respectively.

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Clinical dysregulation of DNA repair by the polynucleotide kinase/phosphatase-XRCC4-DNA ligase IV in neurological disease

Annals of Oncology ◽

10.1093/annonc/mdx361.058 ◽

2017 ◽

Vol 28 ◽

pp. v17

Author(s):

D. Aceytuno ◽

C.G. Piett ◽

Z. Havali-Shahriari ◽

R.A. Edwards ◽

M. Rey ◽

...

Keyword(s):

Dna Repair ◽

Neurological Disease ◽

Dna Ligase ◽

Dna Ligase Iv ◽

Ligase Iv ◽

Polynucleotide Kinase

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The C-Terminal Domain of Cernunnos/XLF Is Dispensable for DNA Repair In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.01521-08 ◽

2008 ◽

Vol 29 (5) ◽

pp. 1116-1122 ◽

Cited By ~ 16

Author(s):

Laurent Malivert ◽

Isabelle Callebaut ◽

Paola Rivera-Munoz ◽

Alain Fischer ◽

Jean-Paul Mornon ◽

...

Keyword(s):

Dna Repair ◽

Nonhomologous End Joining ◽

Dna Ligase ◽

Repair Pathway ◽

End Joining ◽

Functional Assays ◽

The Core ◽

Ligase Iv

ABSTRACT The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4. We demonstrate here, using in vitro-generated Cernunnos mutants and a series of functional assays in vivo, that the C-terminal region of Cernunnos is dispensable for its activity during DNA repair.

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Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1420115112 ◽

2015 ◽

Vol 112 (20) ◽

pp. E2575-E2584 ◽

Cited By ~ 92

Author(s):

Dylan A. Reid ◽

Sarah Keegan ◽

Alejandra Leo-Macias ◽

Go Watanabe ◽

Natasha T. Strande ◽

...

Keyword(s):

Single Molecule ◽

Strand Break ◽

Nonhomologous End Joining ◽

Dna Ligase ◽

End Joining ◽

Dna Ligase Iv ◽

Ligase Iv ◽

End To End

Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.

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