title: A cyclobutane thymine-N4-methylcytosine dimer is resistant to hydrolysis but strongly blocks DNA synthesis.
abstract: Exposure of DNA to ultraviolet light produces harmful crosslinks between adjacent pyrimidine bases, to form cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts. The CPD is frequently formed, and its repair mechanisms have been exclusively studied by using a CPD formed at a TT site. On the other hand, biochemical analyses using CPDs formed within cytosine-containing sequence contexts are practically difficult, because saturated cytosine easily undergoes hydrolytic deamination. Here, we found that N-alkylation of the exocyclic amino group of 2'-deoxycytidine prevents hydrolysis in CPD formation, and an N-methylated cytosine-containing CPD was stable enough to be derivatized into its phosphoramidite building block and incorporated into oligonucleotides. Kinetic studies of the CPD-containing oligonucleotide indicated that its lifetime under physiological conditions is relatively long (∼ 7 days). In biochemical analyses using human DNA polymerase η, incorporation of TMP opposite the N-methylcytosine moiety of the CPD was clearly detected, in addition to dGMP incorporation, and the incorrect TMP incorporation blocked DNA synthesis. The thermodynamic parameters confirmed the formation of this unusual base pair.
authors.affiliation: Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan and Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan.
authors.forename: Junpei
authors.initials: J
authors.lastname: Yamamoto
authors.forename: Tomoko
authors.initials: T
authors.lastname: Oyama
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Kunishi
authors.forename: Chikahide
authors.initials: C
authors.lastname: Masutani
authors.forename: Fumio
authors.initials: F
authors.lastname: Hanaoka
authors.forename: Shigenori
authors.initials: S
authors.lastname: Iwai
mesh_terms: D004247:DNA / Q000096:biosynthesis*; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D011740:Pyrimidine Dimers / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics; D000099270:Y-Family DNA Polymerases
chemical_list: D004247:DNA; D004259:DNA-Directed DNA Polymerase; D009841:Oligonucleotides; D011740:Pyrimidine Dimers; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases