Characterization of simian virus 40 T-antigen double hexamers bound to a replication fork. The active form of the helicase.
Large T-antigen (T-ag) is a viral helicase required for the initiation and elongation of simian virus 40 DNA replication. The unwinding activity of the helicase is powered by ATP hydrolysis and is critically dependent on the oligomeric state of the protein. We confirmed that the double hexamer is the active form of the helicase on synthetic replication forks. In contrast, the single hexamer cannot unwind synthetic forks and remains bound to the DNA as ATP is hydrolyzed. This inability of the T-ag single hexamer to release the DNA fork is the likely explanation for its poor helicase activity. We characterized the interactions of T-ag single and double hexamers with synthetic forks and single-stranded (ss) DNA. We demonstrated that DNA forks promote the formation of T-ag double hexamer. The lengths of the duplex region and the 3' tail of the synthetic forks are the critical factors in assembly of the double hexamer, which is bound to a single fork. We found that the cooperativity of T-ag binding to ss oligonucleotides increased with DNA length, suggesting that multiple consecutive subunits in the hexamer engage the ssDNA.
Alexandrov
Alexander I
AI
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Botchan
Michael R
MR
Cozzarelli
Nicholas R
NR
D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D046911:Macromolecular Substances; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D013539:Simian virus 40 / Q000235:genetics* / Q000276:immunology*
D000952:Antigens, Polyomavirus Transforming; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009711:Nucleotides; D004265:DNA Helicases