Influences of Base Excision Repair Defects on the Lethality and Mutagenicity Induced by Me-lex, a Sequence-selective N3-Adenine Methylating Agent

Abstract

Due to its minor groove selectivity, Me-lex preferentially generates N3-methyladenine (3-MeA) adducts in double-stranded DNA. We undertook a genetic approach in yeast to establish the influence of base excision repair (BER) defects on the processing of Me-lex lesions on plasmid DNA that harbors the p53 cDNA as target. We constructed a panel of isogenic strains containing a reporter gene to test p53 function and the following gene deletions: ⌬mag1, ⌬apn1apn2, and ⌬apn1apn2mag1. When compared with the wild-type strain, a decrease in survival was observed in ⌬mag1, ⌬apn1apn2, and ⌬apn1apn2mag1. The Me-lex-induced mutation frequency increased in the following order: wild type < ⌬mag1< ⌬apn1apn2 ‫؍‬ ⌬apn1apn2mag1. A total of 77 mutants (23 in wild type, 31 in ⌬mag1, and 23 in ⌬apn1apn2) were sequenced. Eighty-one independent mutations (24 in wild type, 34 in ⌬mag1, and 23 in ⌬apn1apn2) were detected. The majority of base pair substitutions were AT-targeted in all strains (14/23, 61% in wild type; 20/34, 59%, in ⌬mag1; and 14/23, 61%, in ⌬apn1apn2). The Mag1 deletion was associated with a significant decrease of GC > AT transitions when compared with both the wild-type and the AP endonuclease mutants. This is the first time that the impact of Mag1 and/or AP endonuclease defects on the mutational spectra caused by 3-MeA has been determined. The results suggest that 3-MeA is critical for Me-lex cytotoxicity and that its mutagenicity is slightly elevated in the absence of Mag1 glycosylase activity but significantly higher in the absence of AP endonuclease activity.Most alkylating agents react with nucleophilic sites on DNA, yielding a complex mixture of DNA lesions (1). Thus, quantitative and qualitative analyses of the biological role(s) of individual DNA lesions and their relative contribution to the mutagenicity and/or toxicity are difficult tasks. Many clinically used anticancer drugs are DNA alkylating agents. Hence, an understanding of which biological effect is caused by a specific DNA lesion may lead to a more rational design of antineoplastic agents.To exercise a significant regulation over the alkylation pattern on DNA, several alkylating agents were recently synthesized, including Me-lex, 1 a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide (lex)

Rev1 and Polζ influence toxicity and mutagenicity of Me-lex, a sequence selective N3-adenine methylating agent

“…Furthermore, the mutation spectra induced by Me-lex in mag1, mag1rad14, and apn1apn2 backgrounds were indistinguishable (p = 0.74, p = 0.85) [2]. Those results were consistent with: (i) unrepaired 3-MeA and AP site in the template strand causing a similar misincorporation by DNA polymerases; and/or (ii) a single common promutagenic lesion, i.e., an AP site, formed from either the enzymatic (Mag1) or hydrolytic release of 3-MeA, being the origin of the mutation [2,21].…”

“…In the present study, the toxicity and mutagenicity of Me-lex induced lesions were evaluated using a p53 functional assay [13][14][15]. Our results are consistent with an involvement of both Polζ and Rev1 in the mutation fixation process of Me-lex induced lesions but with some subtle differences in their relative roles in protecting cells from toxicity and inducing mutations.…”

“…Furthermore, the mutation spectra induced by Me-lex in mag1, mag1rad14, and apn1apn2 backgrounds were indistinguishable (p = 0.74, p = 0.85) [2]. Those results were consistent with: (i) unrepaired 3-MeA and AP site in the template strand causing a similar misincorporation by DNA polymerases; and/or (ii) a single common promutagenic lesion, i.e., an AP site, formed from either the enzymatic (Mag1) or hydrolytic release of 3-MeA, being the origin of the mutation [2,21].TLS DNA polymerases are involved in the mutagenicity of lesions produced by endogenous or exogenous DNA damaging processes [4]. These specialized DNA polymerases exhibit much lower processivity and fidelity compared to replicative DNA polymerases and can insert nucleotides opposite DNA lesions or extend from mispaired bases.…”

“…The in vitro treatment of pTS76 plasmid with Me-lex was performed as previously described [14,15]. Briefly, Melex was dissolved in DMSO immediately before the plasmid pTS76 DNA (3.0 μg) was treated with different Me-lex concentrations (up to 12 mM) in 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 50% EtOH for 1 h at 37 °C.…”

Recent progress on the Ada response for inducible repair of DNA alkylation damage

“…The identification of 3-meA as a cytotoxic rather than a mutagenic DNA lesion has been confirmed by studies using the minor groove-specific alkylating agent Me-lex, a methyl sulphonate ester attached to a dipeptide (Monti et al, 2002). This alkylating agent has a restricted target specificity; it forms 3-meA efficiently, but does not generate adducts in the major groove of DNA, and binds preferentially to A/T rich sequences.…”