Preferential Repair Incision of Cross-Links Versus Monoadducts in Psoralen-Damaged Plasmid DNA by Human Cell-Free Extracts ² Patrick Calsou, ‡ Evelyne Sage, § Ethel Moustacchi, § and Bernard Salles* ,‡ Institut de Pharmacologie et Biologie Structurale, UPR 9062 CNRS, 205 route de Narbonne, 31077 Toulouse Cedex, France, and URA 1292 CNRS, Institut Curie, Section de Recherche, 26 rue d’Ulm, 75231 Paris Cedex 05, France ReceiVed March 25, 1996; ReVised Manuscript ReceiVed July 29, 1996 X ABSTRACT: Upon UVA irradiation psoralens covalently bind to DNA as monoadduct and interstrand cross- link. Psoralen photoadducts are processed Via an excision repair reaction that has been reproduced in Vitro with transcriptionnally active cell-free extracts. A derived in Vitro assay that allows direct quantification of the incised sites has been set up and used to compare the efficiency of the incision reaction on monoadducts and interstrand cross-links. The incision reaction was performed with HeLa cell-free extracts on angelicin or 8-methoxypsoralen (8-MOP)-modified plasmid DNA substrates carrying known amounts of mono- and biadducts, within various relative ratios. In the case of 8-MOP modified plasmids consisting in a mixture of mono- and biadducts on the same DNA molecule, the incision signal was mainly due to the presence of interstrand cross-links. The extent of incision was linear with the number of cross-links up to about 4 cross-links per plasmid and then reached a plateau. The sensitivity of incision defined as the increase of incision by 2-fold over the background level corresponded to about 1 cross-link per plasmid molecule, and about 7% of the total cross-links were repaired under our assay conditions. The incision activity on angelicin monoadducts yielded only 27% when compared to that on 8-MOP cross-links. Furthermore, 8-MOP cross-links lowered the incision extent of angelicin monoadducts when the two photoadducts were present on distinct plasmid DNA molecules. These data are in line with the more rapid excision of psoralen interstrand cross-links Vs monoadducts observed in ViVo. A wide variety of DNA lesions are removed by nucleotide excision repair (NER) 1 which largely contributes to cellular survival. The molecular mechanism is similar from bacteria to humans (Hoeijmakers, 1993a,b; Sancar, 1995). In the current model, the NER process includes two major steps: (i) damage recognition, asymmetric incision of the damaged strand on both sides of the lesion, excision of the damaged oligonucleotide, and (ii) DNA repair synthesis filling in the gap using the complementary strand as template, and ligation. The sequential NER steps have been reproduced in Vitro with damaged plasmid DNA incubated in the presence of tran- scriptionally active cell-free extracts (Sibghat-Ullah et al., 1989; Wood et al., 1988). In this in Vitro assay, the repair activity in protein extracts was measured by the extent of DNA repair synthesis in damaged plasmid as detected by radiolabeled repair patches. It has been estimated that the repair signal is produced by the removal of 1-10% of the lesions contained in plasmid DNA (Sibghat-Ullah et al., 1989; Wood et al., 1988). Despite the use of in Vitro conditions for the repair reaction with cell extracts, NER on plasmid DNA resembles genomic repair since a defective repair capacity has been observed in extracts from repair- deficient Xeroderma pigmentosum (XP) cells belonging to complementation groups from A to G (Hansson et al., 1990, 1991; Reardon et al., 1993; Wood, 1989). Moreover, the complete NER reaction has been reproduced in Vitro with purified proteins (Aboussekhra et al., 1995; Mu et al., 1995; Shivji et al., 1995). In order to determine the size of the DNA repair patch, chemically purified DNA damages have been cloned into plasmid DNA at single site. Using cell-free extracts in an NER reaction, the length of the excised oligonucleotide bearing the lesion, and previously labeled, was determined as 27-29 nucleotides (Huang et al., 1992; Svoboda et al., 1993). The use of substrates carrying unique lesion pointed out a difference in the efficiency in excision of various DNA adducts. For instance, among the cisplatin-induced adducts, the major 1,2 d(GpG) adduct was not (Szymkowski et al., 1992) or was poorly repaired (Huang et al., 1994) although the minor 1,3 d(GpG) lesion was efficiently repaired (O’Donovan et al., 1994). Similarly, among the UVC- induced cyclobutane pyrimidine dimers and 6-4 photoprod- ucts, the latter was repaired while the former was not (Szymkowski et al., 1993a). However, the repair efficiency might be different on a unique lesion of a specific class than on a mixture of these lesions on the same DNA molecule. We addressed the question of a differential repair ef- ficiency in Vitro of a mix of bulky DNA adducts, i.e., monoadducts (MA) and interstrand cross-links (ICL). In order to obtain plasmid DNA modified with a known number of lesions per molecule but differing in their ratio of MA to ICL, we took advantage of the unique photochemistry of psoralen derivatives. Psoralens intercalate into DNA and, upon UVA (320-400 nm) irradiation, undergo photocy- ² This work was supported by grants from Ligue Nationale contre le Cancer, Association pour la Recherche sur le Cancer, and Ministe `re de l’Enseignement Supe ´rieur et de la Recherche. * Corresponding author: Bernard Salles, Fax: (33) 61 17 59 33; E-mail: salles@ipbs.fr. ‡ Institut de Pharmacologie et Biologie Structurale, UPR 9062 CNRS, Toulouse. § URA 1292 CNRS, Institut Curie, Paris. X Abstract published in AdVance ACS Abstracts, November 1, 1996. 1 Abbreviations: NER, nucleotide excision repair; MA, monoadduct; ICL, interstrand cross-link; 8-MOP, 8-methoxypsoralen. 14963 Biochemistry 1996, 35, 14963-14969 S0006-2960(96)00726-X CCC: $12.00 © 1996 American Chemical Society