Deficient DNA end joining activity in extracts from Fanconi anemia fibroblasts

Fanconi anemia (FA) is a genetic disorder associated with genomic instabili ty and cancer predisposition. Cultured cells from FA patients display a hig h level of spontaneous chromosome breaks and an increased frequency of intr agenic deletions, suggesting that FA cells may have deficiencies in properl y processing DNA double strand breaks. In this study, an in vitro plasmid D NA end joining assay was used to characterize the end joining capabilities of nuclear extracts from diploid FA fibroblasts from complementation groups A, C, and D, The Fanconi anemia extracts had 3-9-fold less DNA end joining activity and rejoined substrates with significantly less fidelity than nor mal extracts. Wild-type end joining activity could be reconstituted by mixi ng FA-D extracts with FA-A or FA-C extracts, while mixing FA-A and FA-C ext racts had no effect on end joining activity. Protein expression levels of t he DNA-dependent protein kinase (DNA-PK)/Ku-dependent nonhomologous DNA end -joining proteins Xrcc4, DNA ligase IV, Ku70, and Ku86 in FA and normal ext racts were indistinguishable, as were DNA-dependent protein kinase and DNA end binding activities. The end joining activity as measured by the assay w as not sensitive to the DNA-PK inhibitor wortmannin or dependent on the non homologous DNA end-joining factor Xrcc4, However, when DNA/protein ratios w ere lowered, the end joining activity became wortmannin-sensitive and no di fference in end joining activity was observed between normal and FA extract s. Taken together, these results suggest that the FA fibroblast extracts ha ve a deficiency in a DNA end joining process that is distinct from the DNA- PK/Ku-dependent nonhomologous DNA end joining pathway.