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

Abstract

The relative toxicity and mutagenicity of Me-lex, which selectively generates 3-methyladenine (3-MeA), is dependent on the nature of the DNA repair background. Base Excision Repair (BER) defective S. cerevisiae strains mag1 and apn1apn2 were both significantly more sensitive to Me-lex toxicity, but only the latter is significantly more prone to Me-lex induced mutagenesis. To examine the contribution of translesion synthesis (TLS) DNA polymerases in the bypass of Me-lex-induced lesions, the REV3 and REV1 genes were independently deleted in the parental yeast strain and in different DNA repair deficient derivatives: the Nucleotide Excision Repair (NER) deficient rad14, and the BER deficient mag1 or apn1apn2 strains. The strains contained an integrated ADE2 reporter gene under control of the transcription factor p53. A centromeric yeast expression vector containing the wild-type p53 cDNA was treated in vitro with increasing concentrations of Me-lex and transformed into the different yeast strains. The toxicity of Me-lex induced lesions was evaluated based on the plasmid transformation efficiency compared to the untreated vector, while Me-lex mutagenicity was assessed using the p53 reporter assay. In the present study, we demonstrate that disruption of Polζ (through deletion of its catalytic subunit coded by REV3) or Rev1 (by REV1 deletion) increased Me-lex lethality and decreased Me-lex mutagenicity in both the NER defective (rad14) and BER defective (mag1; apn1apn2) strains. Therefore, Polζ and Rev1 contribute to resistance of the lethal effects of Me-lex induced lesions (3-MeA and derived AP sites) by bypassing lesions and fixing some mutations. KeywordsMe-lex; N3-methyladenine; translesion synthesis; p53; yeast *Corresponding authors: gilberto.fronza@istge.it and goldbi@pitt.edu.. # These authors contributed equally to this work.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. pyrrole-2-carboxamido]-pyrrole-2-carboxamido}propane, is an alkylating agent that preferentially generates N3-methyladenine (3-MeA) adducts in A-T rich regions of double-stranded DNA due to its minor groove selectivity conferred by the lexitropsin dipeptide [1,2]. Me-lex toxicity and mutagenicity are dependent on the DNA repair background. BER defective S. cerevisiae strains that lack 3-methyladenine DNA glycosylase (mag1) or both AP endonucleases (apn1apn2) are significantly and similarly more sensitive to Me-lex toxicity than the parental strain, but only the removal of AP endonuclease activity resulted in a significant increase in mutagenicity. Furthermore, the Me-lex induced mutation spe...

Effect of N3-Methyladenine and an Isosteric Stable Analogue on DNA Polymerization

“…This indicates that the hydrophobic methyl group may sterically block critical interactions with the polymerases, including the Y-family polymerase Pol η . The inability of polymerases to efficiently bypass 3-mA is consistent with the high toxicity and low mutagenicity of Me-lex [15, 16, 32, 37, 38]. The observation that there is a strong sequence-dependency in the bypass of THF within the A 4 sequence by polymerases lacking exonuclease activity, for example, Pol η , may explain the common sequence specific mutation pattern induced by Me-lex in a number of base excision repair backgrounds.…”

Base Excision Repair

“…Most likely, MMR serves as a last major option in repair of genomic uracil. Several translesion bypass polymerases function as backup for the single mammalian DNA glycosylase removing 3-methyladenine, methylpurine DNA glycosylase (MPG, also called AAG) (Johnson et al 2007;Monti et al 2008). These results show extensive backup within and between pathways processing base damage.…”

Starvation Promotes REV1 SUMOylation and p53-Dependent Sensitization of Melanoma and Breast Cancer Cells

“…The p53 tumor suppressor is a key molecule in genomic maintenance and cell fate in mammals (35). Recent studies indicated that p53 and REV1 participate in overlapping cellular processes (36–38). We have sought to study whether REV1 may interact with p53.…”