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 induced lesions on naked plasmid DNA that harbours 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: Dmag1, Dapn1apn2, and Dapn1apn2mag1. All strains contain the ADE2 gene regulated by a p53-responsive promoter to assess p53 transactivation function. In vitro Me-lex damaged plasmid DNA was transformed into the yeast strains in order to determine survivals and p53 mutation frequencies. Compared to the wild type strain, a decrease in survival was observed in Dmag1, Dapn1apn2, and Dapn1apn2mag1. The Me-lex induced mutation frequency increased in the following order: WT < Dmag1< Dapn1apn2 = Dapn1apn2mag1. A total of 77 mutants (23 in WT, 31 in Dmag1 and 23 in Dapn1apn2) were sequenced. Eighty-one independent mutations (24 in WT, 34 in Dmag1, 23 in Dapn1apn2), including a common hotspot at position 602, were detected. The majority of base pair substitutions were AT-targeted in all strains (14/23; 61% in WT, 20/34; 59% in Dmag1, and 14/23, 61% in Dapn1apn2). The Mag1 delation was associated with a significant decrease of GC>AT transitions when compared to 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 and compared. The results suggest that 3-MeA is critical for Me-lex cytotoxicity and that its mutagenicity is only slightly elevated in the absence of Mag1 glycosylase activity, but significantly higher in the absence of AP endonuclease activity.