Mechanism of inactivation of human O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine

Abstract

Human O6-alkylguanine-DNA alkyltransferase was rapidly inactivated by low concentrations of O6-benzylguanine, but the alkyltransferase from the Escherichia coli ogt gene was much less sensitive and alkyltransferases from the E. coli ada gene or from yeast were not affected. O6-Benzyl-2'-deoxyguanosine was less potent than the base, but was still an effective inactivator of the human alkyltransferase and had no effect on the microbial proteins. O6-Allylguanine was somewhat less active, but still gave complete inactivation of both the human and Ogt alkyltransferases at 200 microM in 30 min, slightly affected the Ada protein, and had no effect on the yeast alkyltransferase. O4-Benzylthymidine did not inactivate any of the alkyltransferase proteins tested. Inactivation of the human alkyltransferase by O6-benzylguanine led to the formation of S-benzylcysteine in the protein and to the stoichiometric production of guanine. The rate of guanine formation followed second-order kinetics (k = 600 M-1 s-1). Prior inactivation of the alkyltransferase by reaction with a methylated DNA substrate abolished its ability to convert O6-benzylguanine into guanine. These results indicate that O6-benzylguanine inactivates the protein by acting as a substrate for alkyl transfer and by forming S-benzylcysteine at the acceptor site of the protein. The inability of O6-benzylguanine to inactivate the microbial alkyltransferases may be explained by steric constraints at this site.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoptosis in malignant glioma cells triggered by the temozolomide-induced DNA lesion O6-methylguanine

“…For the time responses, the glioma cells were treated with MNNG and then harvested at the appropriated times. The p53 inhibitor pifithrin-a, which reversibly blocks p53 dependent transcriptional activation (Komarova and Gudkov, 2000), was added 72 h after treatment with either 10 mM MNNG or 100 mM TMZ and apoptosis was determined at time point 144 h. The specific MGMT inhibitor O 6 BG (Pegg et al, 1993) was added 1 h before MNNG treatment at a concentration of 10 mM. The powerful mitogen, Pasteurella multocida toxin (PMT wt ), and the inactive mutant PMT C1165S (Orth et al, 2003) were kept in a 1.1 mg/ml stock solution (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2.5 mM CaCl 2 ) at À201C and used at a concentration of 10 ng/ml (the mitogen was a kind gift of Dr Klaus Aktories, Freiburg).…”

Point mutations at multiple sites including highly conserved amino acids maintain activity, but render O6-alkylguanine‒DNA alkyltransferase insensitive to O6-benzylguanine

“…However, BG is not effective as an inactivator of the AGT proteins from Escherichia coli and yeast [18,23,24]. This is probably due to the steric exclusion of BG from the active-site pocket [14], consisting of the residues shown in Figure 1.…”

“…The mechanism of this inactivation is well understood and involves the recognition of BG as a substrate by the AGT. BG is bound at the active site, where the benzyl group is transferred, releasing guanine and forming S-benzylcysteine in the AGT protein [18]. In animal models, BG has been shown to enhance the cancer chemotherapeutic effects of chloroethylating agents, such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and methylating agents, such as temozolomide [4,19,20].…”

Repair of O6-Benzylguanine by the Escherichia coli Ada and Ogt and the Human O6-Alkylguanine-DNA Alkyltransferases

“…The basis for this inactivation is well understood. The base analog is recognized as a substrate by the human alkyltransferase, and the benzyl group is transferred to the active-site cysteine, forming an S-benzylcysteine adduct and inactivating the protein (10). However, little is known of the means by which O 6 -benzylguanine binds to the active site, and despite the similarity in amino acid sequence, the Ada-C protein was not inactivated by O 6 -benzylguanine (10 -12).…”