Abstract
Fanconi anemia (FA) is a genetic disorder associated with genomic instability and cancer predisposition. Cultured cells from FA patients display a high level of spontaneous chromosome breaks and an increased frequency of intragenic deletions, suggesting that FA cells may have deficiencies in properly processing DNA double strand breaks. In this study, an in vitro plasmid DNA 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 normal extracts. Wild-type end joining activity could be reconstituted by mixing FA-D extracts with FA-A or FA-C extracts, while mixing FA-A and FA-C extracts had no effect on end joining activity. Protein expression levels of the DNA-dependent protein kinase (DNA-PK)/Ku-dependent nonhomologous DNA end-joining proteins Xrcc4, DNA ligase IV, Ku70, and Ku86 in FA and normal extracts were indistinguishable, as were DNA-dependent protein kinase and DNA end binding activities. The end joining activity as measured by the assay was not sensitive to the DNA-PK inhibitor wortmannin or dependent on the nonhomologous DNA end-joining factor Xrcc4. However, when DNA/protein ratios were lowered, the end joining activity became wortmannin-sensitive and no difference in end joining activity was observed between normal and FA extracts. Taken together, these results suggest that the FA fibroblast extracts have a deficiency in a DNA end joining process that is distinct from the DNA-PK/Ku-dependent nonhomologous DNA end joining pathway.
Highlights
Been cloned, FANCA, FANCC, FANCE, FANCF, and FANCG (4 – 8)
Wild-type end joining activity could be reconstituted by mixing Fanconi anemia (FA)-D extracts with FA patients of complementation groups A (FA-A) or FA-C extracts, while mixing FA-A and FA-C extracts had no effect on end joining activity
When DNA/protein ratios were lowered, the end joining activity became wortmanninsensitive and no difference in end joining activity was observed between normal and FA extracts. These results suggest that the FA fibroblast extracts have a deficiency in a DNA end joining process that is distinct from the DNA-PK/Ku-dependent nonhomologous DNA end joining pathway
Summary
To gain further insight into the process of DNA end joining in FA cells, we used an in vitro assay to examine the ability of nuclear protein extracts prepared from diploid FA fibroblasts to rejoin linear plasmid DNA substrates. Nuclear extracts from diploid fibroblasts from patients from complementation groups A, C, and D had 3–9-fold less end joining activity and rejoined linear substrates imprecisely at a higher frequency than extracts from normal donors. This end joining deficiency was not due to the presence of an inhibitor in the FA extracts or to deficiencies in proteins or activities known to be involved in the well characterized DNA-PK/Ku nonhomologous DNA end joining pathway [13]. When a lower substrate DNA/protein ratio was used in the end joining assay, the end joining activity was wortmannin-sensitive, and indistinguishable end joining levels were observed between normal and FA extracts
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