title: Comparison of the biological effects of MMS and Me-lex, a minor groove methylating agent: clarifying the role of N3-methyladenine.
abstract: N3-methyladenine (3-mA), generated by the reaction of methylating agents with DNA, is considered a highly toxic but weakly mutagenic lesion. However, due to its intrinsic instability, some of the biological effects of the adduct can result from the formation of the corresponding depurination product [an apurinic (AP)-site]. Previously, we exploited Me-lex, i.e. {1-methyl-4-[1-methyl-4-(3-methoxysulfonylpropanamido)pyrrole-2-carboxamido]-pyrrole-2 carboxamido}propane, a minor groove equilibrium binder with selectivity for A/T rich sequences that efficiently reacts with DNA to afford 3-mA as the dominant product, to probe the biology of this lesion. Using human p53 cDNA as a target in a yeast system, a weak increase in mutagenicity was observed in the absence of Mag1 (3-methyladenine-DNA glycosylase 1, mag1), the enzyme devoted to remove 3-mA from DNA. Moreover, a significant increase in mutagenicity occurred in the absence of the enzymes involved in the repair of AP-sites (AP endonucleases 1 and 2, apn1apn2). Since methyl methanesulfonate (MMS) has been extensively used to explore the biological effects of 3-mA, even though it produces 3-mA in low relative yield, we compared the toxicity and mutagenicity induced by MMS and Me-lex in yeast. A mutagenesis reporter plasmid was damaged in vitro by MMS and then transformed into wild-type and Translesion Synthesis (TLS) Polζ (REV3) and Polη (RAD30) deficient strains. Furthermore, a mag1rad30 double mutant strain was constructed and transformed with the DNA plasmid damaged in vitro by Me-lex. The results confirm the important role of Polζ in the mutagenic bypass of MMS and Me-lex induced lesions, with Polη contributing only towards the bypass of Me-lex induced lesions, mainly in an error-free way. Previous and present results point towards the involvement of AP-sites, derived from the depurination of 3-mA, in the observed toxicity and mutagenicity.
authors.affiliation: Molecular Mutagenesis and DNA Repair Unit, Istituto di Ricerca e Cura a Carattere Scientifico Azienda Ospedaliera Universitaria San Martino-IST-Istituto Nazionale per la Ricerca sul Cancro, Genoa 16132, Italy. Electronic address: paola.monti@istge.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Monti
authors.affiliation: Molecular Mutagenesis and DNA Repair Unit, Istituto di Ricerca e Cura a Carattere Scientifico Azienda Ospedaliera Universitaria San Martino-IST-Istituto Nazionale per la Ricerca sul Cancro, Genoa 16132, Italy. Electronic address: giorgiafoggetti@libero.it.
authors.forename: Giorgia
authors.initials: G
authors.lastname: Foggetti
authors.affiliation: Molecular Mutagenesis and DNA Repair Unit, Istituto di Ricerca e Cura a Carattere Scientifico Azienda Ospedaliera Universitaria San Martino-IST-Istituto Nazionale per la Ricerca sul Cancro, Genoa 16132, Italy. Electronic address: paola.menichini@hsanmartino.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Menichini
authors.affiliation: Laboratory of Transcriptional Networks, Centre for Integrative Biology, CIBIO, University of Trento, Trento 38060, Italy. Electronic address: inga@science.unitn.it.
authors.forename: Alberto
authors.initials: A
authors.lastname: Inga
authors.affiliation: Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh 15261, PA, USA. Electronic address: goldbi@pitt.edu.
authors.forename: Barry
authors.initials: B
authors.lastname: Gold
authors.affiliation: Molecular Mutagenesis and DNA Repair Unit, Istituto di Ricerca e Cura a Carattere Scientifico Azienda Ospedaliera Universitaria San Martino-IST-Istituto Nazionale per la Ricerca sul Cancro, Genoa 16132, Italy. Electronic address: gilberto.fronza@hsanmartino.it.
authors.forename: Gilberto
authors.initials: G
authors.lastname: Fronza
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000502:physiology; D004259:DNA-Directed DNA Polymerase / Q000502:physiology; D006801:Humans; D008741:Methyl Methanesulfonate / Q000633:toxicity*; D009153:Mutagens / Q000633:toxicity*; D009429:Netropsin / Q000031:analogs & derivatives* / Q000633:toxicity; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology; D000099270:Y-Family DNA Polymerases
chemical_list: D000225:Adenine; D004259:DNA-Directed DNA Polymerase; D008741:Methyl Methanesulfonate; D009153:Mutagens; D009429:Netropsin; D029701:Saccharomyces cerevisiae Proteins; C580784:N3-methyladenine; C110963:methyl lexitropsin; C092602:REV3 protein, S cerevisiae; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases
keywords: 3-m-c3A; 3-mA; 3-methyl-3-deazaadenine; 3-methyladenine-DNA glycosylase 1 defective; AP; AP endonucleases 1 and 2 defective; AP-site; BER; MF; MMS; Me-lex; N3-methyladenine; TLS; Translesion Synthesis; Yeast; apn1apn2; apurinic/apyrimidinic; base excision repair; mag1; methyl methanesulfonate; mutation frequency; sMF; spontaneous mutation frequency; {1-methyl-4-[1-methyl-4-(3-methoxysulfonylpropanamido)pyrrole-2-carboxamido]-pyrrole-2-carboxamido}propane