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N-ethylmaleimide (NEM) modification of a lactose permease mutant containing a single-Cys in place of Ala-122 (helix IV) abolishes active lactose transport. Moreover, lactose, melibiose, and [beta],D-galactopyranosyl 1-thio-[beta],D-galactopyranoside protect against NEM inactivation of lactose transport and/or alkylation of Cys-122 by [[sup.14]C]NEM. Remarkably, however, D-galactose transport is relatively unaffected by NEM, and the monosaccharide affords no protection against NEM inactivation of lactose transport. Consistently, competitive inhibition of [[sup.14]C]galactose transport by lactose, melibiose, or [beta],D-galactopyranosyl 1-thio-[beta],D-galactopyranoside is drastically reduced after NEM modification, whereas inhibition by unlabeled galactose is unaffected. The results indicate that alkylation of Cys-122 selectively inhibits binding and transport of disaccharides, whereas transport of the monosaccharide galactose remains largely unaffected. In addition, although the conservative mutation Ala-122 [right arrow] Ser causes only mild inhibition of lactose transport, the mutations Ala-122 [right arrow] Phe and Ala-122 [right arrow] Tyr lead to marked inhibition. In contradistinction, none of these replacements has a marked effect on galactose transport. The results demonstrate that Ala-122 is a component of the ligand-binding site and provide a strong indication that the side chain at position 122 abuts on the nongalactosyl moiety of D-galactopyranosides. This is in contrast to Cys-148, a neighboring residue in helix V, that interacts with the hydrophobic face of the galactosyl moiety of D-galactopyranosides.

Alkylation of DNA at the [O.sup.6]-position of guanine is one of the most critical events leading to mutation, cancer, and cell death. The enzyme [O.sup.6]-methylguanine-DNA methyltransferase repairs [O.sup.6]-methylguanine as well as a minor methylated base, [O.sup.4]-methylthymine, in DNA. Mouse lines deficient in the methyltransferase (MGMT) gene are hypersensitive to both the killing and to the tumorigenic effects of alkylating agents. We now show that these dual effects of an alkylating agent can be dissociated by introduction of an additional defect in mismatch repair. Mice with mutations in both alleles of the MGMT gene and one of the mismatch repair genes, MLH1, are as resistant to methylnitrosourea (MNU) as are wild-type mice, in terms of survival, but do have numerous tumors after receiving MNU. In contrast to [MGMT.sup.-/-] [MLH1.sup.+/+] mice with decrease in size of the thymus and hypocellular bone marrow after MNU administration, no conspicuous change was found in [MGMT.sup.-/-] [MLH1.sup.-/-] mice treated in the same manner. Thus, killing and tumorigenic effects of an alkylating agent can be dissociated by preventing mismatch repair pathways.

Alkylation of DNA by 7r,8t-dihydroxy,9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) forms mainly trans adducts (with respect to the C-9/10 positions). We recently described a halide-catalyzed pathway that preferentially generates cis adducts and now report that the trans chlorohydrin of anti-BPDE (trans-BPDCH) is an intermediate in the chloride-catalyzed reaction. trans-BPDCH was synthesized, and both it and anti-BPDE were reacted with deoxyadenosine as a model DNA nucleophile. The stereochemistry and yields of deoxyadenosine adducts were determined as a function of chloride concentration. In the absence of salt, the fraction of cis adducts obtained from anti-BPDE and trans-BPDCH are 0.33 and 0.67, respectively. Adding sodium chloride increases the fraction of cis adducts (and consequently decreases the fraction of trans adducts), with the midpoint of the increase for both substrates at approximately 35-40 mM chloride. The chloride-dependent curves for BPDE and BPDCH converge at 1 M chloride, where the fraction of cis adducts is 0.88. Chloride also increases the total yield of cis adducts with either substrate, whereas the yield of trans adducts from the chlorohydrin is not significantly changed. These results support a mechanism by which chloride ion undergoes nucleophilic addition to the benzylic C-10 position of anti-BPDE. This generates a trans halohydrin that alkylates DNA with inversion of configuration to form a cis adduct. This pathway may have biological significance because chlorohydrins could form in serum or in cells with relatively high intracellular concentrations of chloride.

The role of the mutagenic lesion O-6-MeGua in the neoplastic transformation arising from the biological effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated. Subclones of the 10T1/2 cell line expressing the bacterial DNA repair genes ada and ogt, whose products transfer the alkyl group from the O-6 position to cysteine residues, were developed. The results showed that the O-6-alkylguanine-DNA alkyltransferase product of the ada or the ogt genes can protect against MNNG-induced mutation and neoplastic transformation. However, O-6-MeGua is a major but not unique factor in MNNG-induced neoplastic transformation.

A study was done on the effect of purified human 1,N6-ethenoadenine-binding protein and purified human 3-methyladenine-DNA glycosylase on 1,N6-ethenoadenine and 3-methyladenine. It was shown that the protein was a glycosylase which releases 1,N6-ethenoadenine and 3-methyladenine from methylated DNA. In addition, the 3-methyladenine-DNA was found to act in the same manner and binds to ethenoadenine-containing oligonucleotide cleaving both ethenoadenine and 3-methyladenine from DNA containing the adducts.