title: 3-Methyl-3-deazaadenine, a stable isostere of N3-methyl-adenine, is efficiently bypassed by replication in vivo and by transcription in vitro.
abstract: The goal of the present work was to determine the impact of N3-methyladenine (3-mA), an important lesion generated by many environmental agents and anticancer drugs, on in vivo DNA replication and in vitro RNA transcription. Due to 3-mA chemical instability, the stable isostere 3-methyl-3-deazaadenine (3-m-c(3)A) was site specifically positioned into an oligodeoxynucleotide. The oligomer was, then incorporated into a vector system that is rapidly converted to ssDNA inside yeast cells and requires DNA replication opposite the lesion for plasmid clonal selection. For control purposes, an adenine or a stable apurinic/apyrimidinic (AP)-lesion was placed at the same site. The presence of each lesion in the oligonucleotide was confirmed by MALDI-TOF analysis. Plasmids were then transfected into yeast cells. While the AP-site dramatically reduced plasmid replication in all strains, the 3-m-c(3)A had a slight effect in the rad30 background which significantly increased only in a rev3rad30 background. Considering TLS events opposite 3-m-c(3)A, the lack of Polη was associated with a substantial increase in AT>GC transitions (p=0.0011), while in the absence of Polζ only events derived from an error free bypass were detected. The 3-m-c(3)A also did not affect in vitro transcription, while the AP-site was a strong block to T7 RNA progression when located in the transcribed strand. We conclude that, in these experimental systems, 3-m-c(3)A is efficiently bypassed by replication in vivo and by transcription in vitro.
authors.affiliation: Molecular Mutagenesis and DNA Repair Unit, Department of Epidemiology and Prevention, National Cancer Research Institute (IST), 16132 Genova, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Monti
authors.forename: Christopher
authors.initials: C
authors.lastname: Broxson
authors.forename: Alberto
authors.initials: A
authors.lastname: Inga
authors.forename: Ruo-wen
authors.initials: RW
authors.lastname: Wang
authors.forename: Paola
authors.initials: P
authors.lastname: Menichini
authors.forename: Silvia
authors.initials: S
authors.lastname: Tornaletti
authors.forename: Barry
authors.initials: B
authors.lastname: Gold
authors.forename: Gilberto
authors.initials: G
authors.lastname: Fronza
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism; D001080:Apurinic Acid / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism*; D004261:DNA Replication; D004271:DNA, Fungal / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics; D055786:Gene Knockout Techniques; D017930:Genes, Reporter; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics; D000099270:Y-Family DNA Polymerases
chemical_list: D000225:Adenine; D001080:Apurinic Acid; D018736:DNA Adducts; D004271:DNA, Fungal; D004259:DNA-Directed DNA Polymerase; D012321:DNA-Directed RNA Polymerases; D029701:Saccharomyces cerevisiae Proteins; D014764:Viral Proteins; C528907:3-deaza-3-methyladenine; C062210:bacteriophage T7 RNA polymerase; C092602:REV3 protein, S cerevisiae; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases