Site-dependent inhibition by single O6-methylguanine bases of SV40 T-antigen interactions with the viral origin of replication.
The effects of O6-methylguanine on the reactions involved in initiation of DNA replication were investigated by measuring the interactions of SV40 T antigen with oligonucleotides substituted with the methylated base. O6-Methylguanine residues were positioned in either binding site I or binding site II of the SV40 origin of replication. Binding of purified T antigen, measured by both nitrocellulose filter binding and delayed oligonucleotide migration, was unaffected by the presence of seven methylated bases in binding site II. Single substitutions within binding site I were sufficient to inhibit T-antigen binding, and the extent of inhibition was dependent on the position of O6-methylguanine in the DNA sequence. Unwinding by T antigen was analyzed by measuring displacement of a single-stranded oligonucleotide from similarly substituted, partially duplex substrates. The presence of three O6-methylguanine residues in binding site I facilitated the helicase activity of T antigen. In contrast, single O6-methylguanine bases inhibited unwinding. A correlation was observed between the position of the methylated base and the inhibition of both binding and unwinding by T antigen.
Bignami
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Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, U.K.
Karran
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Lane
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D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D004279:DNA, Viral / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D013539:Simian virus 40 / Q000187:drug effects / Q000235:genetics* / Q000276:immunology
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D006147:Guanine; C008449:O-(6)-methylguanine; D004265:DNA Helicases