The hypersensitivity of Fanconi anemia, complementation group A, (FA-A) cells to agents which produce DNA interstrand cross-links correlates with a defect in their ability to repair this type of damage. In order to more clearly elucidate this repair defect, chromatin-associated protein extracts from FA-A cells were examined for ability to endonucleolytically produce incisions in DNA at sites of interstrand cross-links. A defined 140 bp DNA substrate was constructed with a single site-specific monoadduct or interstrand cross-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 produce dual incisions in DNA at sites of interstrand cross-links. Specifically, there is defective incision on the 3′- and 5′-sides of both the furan and pyrone sides of the cross-link. This defect is corrected in FA-A cells transduced with a retroviral vector expressing FANCA cDNA. At the site of a TMP monoadduct, FA-A cells can introduce incisions on both the 3′- and 5′-sides of the furan side monoadduct, but are defective in ability to produce these incisions on the pyrone side monoadduct. These studies also indicate that 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 interstrand cross-links could be related to reduced levels of non-erythroid α spectrin (αSpIIΣ*) in the extracts from FA-A cells. αSpIIΣ* could act as a scaffold to align proteins involved in cross-link repair and enhance their interactions; a deficiency in αSpIIΣ* 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.
Keywords: FA, Fanconi anemia; FA-A, Fanconi anemia, complementation group A; HMT, 4'-hydroxymethyl-4,5′,8-trimethylpsoralen; NER, nucleotide excision repair; αSpIIΣ*, non-erythroid α spectrin; TMP, 4,5′,8-trimethylpsoralen; UDS, unscheduled DNA synthesis; XPF, xeroderma pigmentosum complementation group F.
Journal Article. 8769 words. Illustrated.
Subjects: Clinical Cytogenetics and Molecular Genetics
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