Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV.
BACKGROUND
Mammalian cells deficient in the XRCC4 DNA repair protein are impaired in DNA double-strand break repair and are consequently hypersensitive to ionising radiation. These cells are also defective in site-specific V(D)J recombination, a process that generates the diversity of antigen receptor genes in the developing immune system. These features are shared by cells lacking components of the DNA-dependent protein kinase (DNA-PK). Although the XRCC4 gene has been cloned, the function(s) of XRCC4 in DNA end-joining has remained elusive.


RESULTS
We found that XRCC4 is a nuclear phosphoprotein and was an effective substrate in vitro for DNA-PK. Human XRCC4 associated extremely tightly with another protein(s) even in the presence of 1 M NaCl. Co-immunoprecipitation and adenylylation assays demonstrated that this associated factor was the recently identified human DNA ligase IV. Consistent with this, XRCC4 and DNA ligase IV copurified exclusively and virtually quantitatively over a variety of chromatographic steps. Protein mapping studies revealed that XRCC4 interacted with ligase IV via the unique carboxy-terminal ligase IV extension that comprises two tandem BRCT (BRCA1 carboxyl terminus) homology motifs, which are also found in other DNA repair-associated factors and in the breast cancer susceptibility protein BRCA1.


CONCLUSIONS
Our findings provide a function for the carboxy-terminal region of ligase IV and suggest that BRCT domains of other proteins may mediate contacts between DNA repair components. In addition, our data implicate mammalian ligase IV in V(D)J recombination and the repair of radiation-induced DNA damage, and provide a model for the potentiation of these processes by XRCC4.
Critchlow
S E
SE
Wellcome/CRC Institute, Cambridge, UK.
Bowater
R P
RP
Jackson
S P
SP
D000818:Animals; D019313:BRCA1 Protein / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000302:isolation & purification / Q000378:metabolism*; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008954:Models, Biological; D009687:Nuclear Proteins; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D013379:Substrate Specificity
D019313:BRCA1 Protein; D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; D009687:Nuclear Proteins; C101467:XRCC4 protein, human; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D011088:DNA Ligases; D000072481:DNA Ligase ATP