Preferential binding of simian virus 40 T-antigen dimers to origin region I.
The sequence components that direct high-affinity binding of simian virus 40 (SV40) T antigen to SV40 origin region I are composed of two recognition pentanucleotides separated by a spacer. This region has binding sites for two T-antigen monomeric units. We extended the tripartite region I sequence by one and two sets of spacers and pentanucleotides and also shortened the region by one pentanucleotide. Our T-antigen-binding studies with these constructs show that the protein has a strong preference for binding to an even rather than an odd number of pentanucleotides separated by spacer sequences. Gel retardation assays reveal that the size of the complex formed between the 17-base-pair region I sequence and T antigen did not increase when the sequence was extended with one spacer-pentanucleotide sequence but did increase with two such units. DNase I footprinting and fragment assay experiments indicate that the protein did not protect a pentanucleotide that was not paired with another pentanucleotide. The unpaired pentanucleotide resumed its binding activity when it was paired with a spacer and another pentanucleotide sequence. We propose that T antigen binds to region I as a preformed dimer.
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Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
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D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D003850:Deoxyribonuclease I; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D013539:Simian virus 40 / Q000235:genetics / Q000276:immunology*
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D003850:Deoxyribonuclease I