O6-methylguanine in the SV40 origin of replication inhibits binding but increases unwinding by viral large T antigen.
To study the effect of the potentially cytotoxic base O6-methylguanine (O6-meG) on the initiation of DNA replication, double-stranded oligonucleotides corresponding to the SV40 origin of replication were constructed in which O6-meG replaced guanine in one strand. Out of 14 methylated residues, 10 were present in the Binding sites for T antigen (3 in Binding Site 1 and 7 in Binding Site 2). Binding of purified T antigen to the substituted oligonucleotide was considerably reduced in comparison to the unsubstituted one, as measured by nitrocellulose filter binding. Both the ATP-dependent and ATP-independent binding of T antigen were affected by the presence of the methylated base. Band shift analysis revealed an altered pattern of delayed-migrating complexes of T antigen with the O6-meG-containing oligonucleotide. Competition experiments, in which unmodified oligonucleotides containing Binding Site 1 or 2 were included in the binding assays, indicated that the affinity of T antigen for the O6-meG modified sites was reduced. When partially duplex oligonucleotides containing either Binding Site 1 or Site 2 of the origin of replication were used as substrates for the helicase activity of T antigen, the presence of O6-meG increased the extent of T antigen catalysed displacement of single-stranded DNA fragments.
Bignami
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Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, UK.
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000956:Antigens, Viral / Q000378:metabolism; D000957:Antigens, Viral, Tumor / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D008969:Molecular Sequence Data; D013539:Simian virus 40 / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D014779:Virus Replication
D000956:Antigens, Viral; D000957:Antigens, Viral, Tumor; D004279:DNA, Viral; D006147:Guanine; D000255:Adenosine Triphosphate; C008449:O-(6)-methylguanine; D004265:DNA Helicases