Repair of DNA interstrand cross-links is a complex process critical to which is the identification of sites of damage by specific proteins. We have recently identified the structural protein nonerythroid alpha spectrin (alphaSpIISigma) as a component of a nuclear protein complex in normal human cells which is involved in the repair of DNA interstrand cross-links and have shown that it forms a complex with the Fanconi anemia proteins FANCA, FANCC, and FANCG. Using DNA affinity chromatography, we now show that alphaSpIISigma, present in HeLa cell nuclei, specifically binds to DNA containing psoralen interstrand cross-links and that the FANCA, FANCC, and FANCG proteins are bound to this damaged DNA as well. That spectrin binds directly to the cross-linked DNA has been shown using purified bovine brain spectrin (alphaSpIISigma1/betaSpIISigma1)2. Binding of the Fanconi anemia (FA) proteins to the damaged DNA may be either direct or indirect via their association with alphaSpIISigma. These results demonstrate a role for alpha spectrin in the nucleus as well as a new function for this protein in the cell, an involvement in DNA repair. alphaSpIISigma may bind to cross-linked DNA and act as a scaffold to help in the recruitment of repair proteins to the site of damage and aid in their alignment and interaction with each other, thus enhancing the efficiency of the repair process.

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 alpha spectrin (alphaSpIISigma*) in the extracts from FA-A cells. alphaSpIISigma* could act as a scaffold to align proteins involved in cross-link repair and enhance their interactions; a deficiency in alphaSpIISigma* 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.

Repair of DNA interstrand cross-links is a multistep process, critical to which is production of incisions at the site of the lesion resulting in the unhooking of the cross-link from DNA. We have previously shown that XPF is involved in production of incisions at the site of a psoralen interstrand cross-link and that in Fanconi anemia, complementation group A (FA-A) cells, there is a deficiency in these incisions. We now demonstrate that in FA complementation group B, C, D2, F, and G cells there is also a deficiency in production of these incisions. Involvement of FA proteins in this process is demonstrated by the ability of FA cells, corrected with the appropriate FANC cDNAs, to produce these incisions and by inhibition of these incisions by antibodies against these proteins. This incision deficiency correlates with reduced levels of DNA repair synthesis in these cells and is not due to reduced levels of XPF. FA proteins could be influencing this incision process by interacting either with proteins involved in the unhooking step or with damaged DNA, acting as a damage sensor. The results also demonstrate that FA cells are undergoing apoptosis by 12 h after interstrand cross-link damage. It is thus proposed that the single-strand breaks known to be created in DNA during apoptosis could mask the deficiency in ability of FA cells to incise cross-linked DNA and could account for the reported discrepancy as to whether FA cells are deficient in the incision step of the repair process.

Human chromatin-associated protein extracts were examined for endonucleolytic activity on a defined 132-base pair DNA substrate containing a single, site-specific 4,5,8-trimethylpsoralen plus long wavelength ultraviolet light-induced furan side or pyrone side monoadduct or interstrand cross-link. These extracts produced incisions on both the 3 and 5 sides of each of these lesions. The distance between the 3 and 5 incisions at sites of a furan side monoadduct or cross-link was 9 nucleotides, and at sites of a pyrone side monoadduct or cross-link it was 17 nucleotides. Incisions on the 3 side of both types of furan side and pyrone side adducts were similar and were either at the fourth or fifth phosphodiester bond from the adducted thymine, depending upon the adduct. However, greater differences were observed between sites of 5 incision. This incision occurred at the fifth and sixth phosphodiester bonds from the adducted thymine at sites of furan side monoadducts and cross-links, respectively, and at the 13th and 14th phosphodiester bonds at sites of pyrone side monoadducts and cross-links, respectively. Thus, direct analysis of sites of endonucleolytic incision reveals that the location of sites of incision on TMP-adducted substrates depends upon the type of adduct present.Repair of DNA interstrand cross-links is critical for a number of cellular processes such as transcription and DNA replication and therefore is particularly important for the maintenance of genetic integrity and cellular survival. A nucleotide excision repair mechanism is responsible in both prokaryotes and eukaryotes for the removal of this type of lesion (1-3). Although the molecular basis of repair of DNA interstrand cross-links has been extensively studied in bacteria, the proteins involved in the removal of these lesions in mammalian cells and their mechanism of action is largely unknown. A critical, rate-limiting step in this repair process is the initial damage recognition and incision step in which a protein specifically locates or recognizes a site of damage and an endonuclease makes an incision on the DNA strand at or near this site. We have recently identified a damage recognition protein in normal human chromatin, which binds to DNA containing interstrand cross-links produced by 4,5Ј,8-trimethylpsoralen (TMP) 1 plus long wavelength ultraviolet (UVA) light, and our studies suggest that it plays a role in the repair of interstrand cross-links (4). However, an endonuclease that specifically incises DNA at sites of these interstrand cross-links has heretofore not been isolated from mammalian cells.A number of different agents have been shown to produce interstrand cross-links in DNA. One of the most definitive of these, whose reaction with DNA has been well characterized, is psoralen plus UVA light. Psoralens are a group of three-ring heterocyclic furocoumarins that contain two reactive double bonds, a 4Ј,5Ј-double bond in the furan ring and a 3,4-double bond in the pyrone ring (5-8). Psoralen-DNA adducts are formed in three stages; pso...

XPF forms a heterodimeric complex with ERCC1 and is required for the repair of DNA interstrand cross-links. In association with ERCC1, it is involved in production of the 5' incision at the site of a psoralen interstrand cross-link as well as the 3' incision. The present study was carried out to determine the functional domains of XPF that are important in the production of the 5' and 3' incisions that occur at a site of a psoralen interstrand cross-link. Monoclonal antibodies (mAbs) were utilized that had been generated against polypeptide fragments of XPF and affinity-mapped to specific regions of XPF. These mAbs were examined for their ability to differentially inhibit production of dual incisions in DNA by normal human chromatin-associated protein extracts that contain XPF and ERCC1. These studies show that two regions of XPF, one N-terminal region from amino acids 12-166 and one C-terminal region from amino acids 702-854, are the most important in the production of the 5' incision. The same N-terminal region and the C-terminal region from amino acids 702-916 are also involved in the 3' incision, though to a much lesser extent. Since this C-terminal region corresponds to the proposed site of interaction of ERCC1 with XPF, these results suggest that binding of ERCC1 to XPF is critical for its ability to produce the 5' and 3' incisions at the site of an interstrand cross-link, possibly through activation or regulation of the endonucleolytic activity of the N-terminal domain of XPF.