Abstract
O6-alkylguanine-DNA alkyltransferase (AGT) is a single-cycle DNA repair enzyme that removes pro-mutagenic O6-alkylguanine adducts from DNA. Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood. Here, we examine the functions of this enzyme on O6-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex. On a folded 22-nt G-quadruplex substrate, binding saturated at 2 AGT:DNA, significantly less than the ∼5 AGT:DNA found with linear single-stranded DNAs of similar length, and less than the value found with the telomere sequence under conditions that inhibit quadruplex formation (4 AGT:DNA). Despite these differences, AGT repaired 6mG adducts located within folded G-quadruplexes, at rates that were comparable to those found for a duplex DNA substrate under analogous conditions. Repair was kinetically biphasic with the amplitudes of rapid and slow phases dependent on the position of the adduct within the G-quadruplex: in general, adducts located in the top or bottom tetrads of a quadruplex stack exhibited more rapid-phase repair than did adducts located in the inner tetrad. This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs.
Highlights
Cellular DNA is exposed to many chemical and physical agents that can modify DNA bases and/or backbone structures
Taken with evidence that G-quadruplexes are substantially unfolded in the absence of K+ (Table 2), these results suggest that the fast phase of repair may correspond to a fraction of 6mG residues that is immediately available for interaction with AGT, while the slow phase may correspond to residues that become available as a result of a conformational shift in DNA or protein
AGT binds and repairs O6-alkylguanine lesions in singlestranded and duplex DNAs with little difference in affinity or alkyltransfer rate [13,15]. These modest differences, for substrates that differ in secondary structure, charge density, local stiffness and base-stacking stabilities [43] led us to ask whether AGT could function on substrates with other secondary structures
Summary
Cellular DNA is exposed to many chemical and physical agents that can modify DNA bases and/or backbone structures. One particular type of damage is the alkylation of O6-atoms in guanine residues. This modification is cytotoxic and mutagenic in cells and carcinogenic in model organisms [1,2,3]. O6-alkylguanines are repaired by the O6-alkylguanine DNA alkyltransferase (AGT, known as methylguanine methyltransferase, MGMT) [4,5]. This enzyme protects tumor cells against chemotherapeutic DNA-alkylating drugs [5,6,7,8]. AGT inhibitors are currently in clinical trial with the aim of improving the efficacy of alkylating agents in cancer chemotherapy [9,10,11]
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