Open Access8-Hydroxyadenine (7, 8-dihydro-8-Oxoadenine) induces misincorporation inin vitroDNA synthesis and mutations in NIH 3T3 cells
Author(s) -
Hiroyuki Kamiya,
Hiroyuki Miura,
Naoko MurataKamiya,
Hiroyuki Ishikawa,
Tomoki Sakaguchi,
Hideo Inoue,
Tomomi Sasaki,
Chlkahide Masutanl,
Fumio Hanaoka,
Susumu Nishimura,
Eiko Ohtsuka
Publication year - 1995
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/23.15.2893
Subject(s) - biology , microbiology and biotechnology , dna polymerase , klenow fragment , dna polymerase ii , dna polymerase i , dna clamp , polymerase , dna , dna synthesis , transversion , gene , point mutation , transfection , mutation , biochemistry , polymerase chain reaction , exonuclease , reverse transcriptase
An oligodeoxyribonucleotide containing 8-hydroxyadenine (OH8Ade) was chemically synthesized and single- and double-stranded c-Ha-ras gene fragments with OH8Ade at the second position of codon 61 were prepared. The single-stranded ras gene fragment was used as a template for in vitro DNA synthesis with the Klenow fragment of Escherichia coli DNA polymerase I, Taq DNA polymerase, rat DNA polymerase beta and mouse DNA polymerase alpha. The former two enzymes exclusively incorporated dTMP opposite OH8Ade. The DNA polymerases alpha and beta misinserted dGMP, and dAMP and dGMP, respectively. The c-Ha-ras gene was constructed using the double-stranded ras gene fragment containing OH8Ade and was transfected into NIH 3T3 cells. The gene with OH8Ade induced focus formation, indicating that OH8Ade elicited point mutations in cells. When c-Ha-ras genes present in transformed cells were analyzed, an A-->G transition and an A-->C transversion were detected. These results indicate that OH8Ade induced misincorporation in in vitro DNA synthesis and mutations in mammalian cells.