Fanconi anemia, complementation group A, cells are defective in ability toproduce incisions at sites of psoralen interstrand cross-links

The hypersensitivity of Fanconi anemia, complementation group A, (FA-A) cel ls to agents which produce DNA interstrand cross-links correlates with a de fect in their ability to repair this type of damage. In order to more clear ly elucidate this repair defect, chromatin-associated protein extracts from FA-A cells were examined for ability to endonucleolytically produce incisi ons in DNA at sites of interstrand cross-links. A defined 140 bp DNA substr ate was constructed with a single site-specific monoadduct or interstrand c ross-link produced by 4,5',8-trimethylpsoralen (TMP) plus long wavelength ( UVA) light. Our results show that FA-A cells are defective in ability to pr oduce dual incisions in DNA at sites of interstrand cross-links. Specifical ly, there is defective incision on the 3'- and 5'-sides of both the furan a nd pyrone sides of the crosslink. This defect is corrected in FA-A cells tr ansduced with a retroviral vector expressing FANCA cDNA, At the site of a T MP monoadduct, FA-A cells can introduce incisions on both the 3'- and 5'-si des of the furan side monoadduct, but are defective in ability to produce t hese incisions on the pyrone side monoadduct. These studies also indicate t hat XPF is involved in production of the 5' incision by the normal extracts on these substrates. These results correlate with our previous work, which showed that FA-A cells are mainly defective in ability to repair psoralen interstrand cross-links with a lesser defect in ability to repair psoralen monoadducts. This defect in endonucleolytic incision at sites of TMP inters trand cross-links could be related to reduced levels of non-erythroid a spe ctrin (alpha SpII Sigma*) in the extracts from FA-A cells, alpha SpII Sigma * could act as a scaffold to align proteins involved in cross-link repair a nd enhance their interactions; a deficiency in alpha SpII Sigma* could thus lead to reduced efficiency of repair and the decreased levels of incisions we observe at sites of interstrand cross-links in FA-A cells.