INTERACTIONS OF HUMAN O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE (AGT) WITH SHORT DOUBLE-STRANDED DNAS

O6-alkylguanine and O4-alkylthymine are mutagenic and carcinogenic adducts that are formed in DNA as a result of exposure to alkylating agents [1–3]. Both adducts are repaired by the ubiquitous repair protein, O6-alkylguanine-DNA alkyltransferase (AGT)1 [1,4,5]. Ironically, AGT also protects cells against chemotherapeutic drugs that methylate or chloroethylate DNA [6, 7]. Clinical trials of AGT-inhibitors are underway in an attempt to increase the efficacy of such drugs in cancer chemotherapy [8–10]. In addition, overexpression of AGT in hematopoietic stem cells is under study as a means to protect normal cells against the myelosuppressive effects of alkylating-agent chemotherapy [10, 11]. In spite of the interest focused on AGT as a result of its relevance to cancer, relatively little is known about its mechanisms of interaction with DNA and with proteins in its environment. A more complete characterization of these interactions is justified by the probability that they influence the repair activities of AGT in vivo as well as our ability to control DNA repair for therapeutic purposes. Human AGT is a monomeric protein (207 amino acids, Mr = 21,519) that is expressed constitutively in normal cells [3, 5, 7] but may be overexpressed in some tumors that have been exposed to alkylating agents [12]. The protein binds DNA with modest affinity and little sequence specificity [13–15]. Studies with repair-inactive mutant proteins and with proteins in which the alkyltransfer function has been inactivated by prior alkylation of residue C145 (described below) suggest that binding affinities for alkylated DNA sites are only marginally greater than for the corresponding non-alkylated sequences ([15], Fried, unpublished results). While these results do not rule out preferential binding to O6-alkylguanine or O4-alkylthymine containing sequences, they raise the intriguing possibility that the lesion-search mechanism of AGT may not depend strongly on preferential binding. In repair reactions, each protein molecule mediates the transfer of a single alkyl group from the O6 position of guanine or O4 position of thymine to an active-site cysteine (C145 in the human protein). This reaction returns the DNA base to an unmodified state and permanently inactivates the alkyl-acceptor activity of the enzyme, which is ultimately degraded [4, 16]. As a result of this stoichiometric mechanism, the number of adducts that can be repaired at one time depends on the steady-state cellular concentration of the non-alkylated form of AGT [4, 5] and on its ability to partition between lesion sites and the large excess of genomic DNA in which they are embedded. Several crystal structures of AGT-DNA complexes have been obtained in recent years. Those determined by Daniels et al. (PDB numbers 1T38 and 1T39) contain 1:1 AGT-DNA complexes [17] while that of Duguid et al. (PDB number 1YFH) contains two AGT molecules making separate contact with a single DNA molecule and a third binding a second DNA [18]. The isolated binding of single protein molecules that is a recurring theme in these structures contrasts with the close-packed, cooperative pattern of binding observed in free solution with both single-stranded and duplex templates ([15, 19], this work). These differences in binding mode suggest that binding in free solution may involve a different ensemble of molecular contacts than is present in the crystal structures. A more complete characterization of cooperative AGT-DNA interactions may allow us to account for this difference.