title: Excision of 8-methylguanine site-specifically incorporated into oligonucleotide substrates by the AlkA protein of Escherichia coli.
abstract: 8-Methyl-2'-deoxyguanosine (8-medGuo) has been shown to be a major stable alkylation product of 2'-deoxyguanosine induced by methyl radical attack on DNA. Moreover, by using primer extension assays, the latter DNA modification has recently been reported to be a miscoding lesion by generating G to C and G to T transversions and deletions in vitro. However, no data have been reported up to now, concerning the processing of this C8-alkylated nucleoside by the DNA repair machinery. Therefore, we have investigated the capability of excision of 8-methylguanine (8-meGua) site specifically incorporated into oligonucleotide substrates by several bacterial, yeast and mammalian DNA N-glycosylases. The results show that the 3-methyladenine (3-meAde) DNA glycosylase II (AlkA protein) from Escherichia coli is the only DNA N-glycosylase tested able to remove 8-meGua from double-stranded DNA fragments. Moreover, the activity of AlkA for 8-meGua varied markedly depending on the opposite base in DNA, being the highest with Adenine and Thymine and the lowest with Cytosine and Guanine. The removal of 8-meGua by AlkA protein was compared to that of 7-methylguanine (7-meGua) and hypoxanthine (Hx). The rank of damage as a substrate for AlkA being 7-meGua>8-meGua>Hx. In contrast, the human 3-meAde DNA N-glycosylase (Mpg) is not able to release 8-meGua paired with any of the four DNA bases. We also show that, DNA N-glycosylases involved in the removal of oxidative damage, such as Fpg or Nth proteins from E. coli, Ntg1, Ntg2 or Ogg1 proteins of Saccharomyces cerevisiae, or human Ogg1 do not release 8-meGua placed opposite any of the four DNA bases. Furthermore, HeLa and Chinese hamster ovary (CHO) cell free protein extracts do not show any cleavage activity at 8-meGua paired with adenine or cytosine, which suggests the absence of base excision repair (BER) of this lesion in mammalian cells.
authors.affiliation: Laboratoire des Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR 5046 CEA-CNRS-UJF, Département de Recherche Fondamentale sur la Matière Condensée, CEA-Grenoble, 17 Avenue des Martyrs F-38054 Cedex 9, Grenoble, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Gasparutto
authors.forename: Claudine
authors.initials: C
authors.lastname: Dhérin
authors.forename: Serge
authors.initials: S
authors.lastname: Boiteux
authors.forename: Jean
authors.initials: J
authors.lastname: Cadet
mesh_terms: D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D020137:Base Pair Mismatch; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003596:Cytosine / Q000737:chemistry; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics; D045647:DNA Glycosylases; D017931:DNA Primers; D004260:DNA Repair / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology*; D005092:Exonucleases / Q000494:pharmacology; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; C066771:8-methylguanine; D006147:Guanine; D003596:Cytosine; D004247:DNA; D005092:Exonucleases; D000469:Alkaline Phosphatase; D015719:Single-Strand Specific DNA and RNA Endonucleases; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C076131:DNA-3-methyladenine glycosidase II