Characterization of Simian Virus 40 T-antigen Double Hexamers Bound to a Replication Fork: THE ACTIVE FORM OF THE HELICASE

Alexander I Alexandrov,Michael R Botchan,Nicholas R Cozzarelli

doi:10.1074/jbc.m207022200

Alexander I Alexandrov, Michael R Botchan + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m207022200

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Abstract

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.

Highlights

DNA helicases are ubiquitous enzymes that function as cellular motors to unwind DNA duplexes at the expense of NTP hydrolysis [1]
Binding of T-ag to single-stranded DNA (ssDNA)—As binding of T-ag to synthetic forks is expected to occur, at least in part, through interactions with the ssDNA tails, we began our study by investigating the length dependence of T-ag binding to ssDNA using gel-shift analyses (Fig. 1A)
The pattern of DNA binding by T-ag is complex, as the binding is linked to the assembly of higher order oligomeric structures of T-ag

Summary

THE ACTIVE FORM OF THE HELICASE*

We confirmed that the double hexamer is the active form of the helicase on synthetic replication forks. The single hexamer cannot unwind synthetic forks and remains bound to the DNA as ATP is hydrolyzed. Translocation involves a series of binding and release events during which helicases cycle between DNA binding states varying in affinity using steps in the NTP hydrolysis cycle as “switches.” Helicases translocate unidirectionally along ssDNA.1 This directionality is intrinsic to their unwinding activity and is revealed by a tight association to only one of the product single strands. To construct a model for how T-ag interacts with the replication forks during unwinding, we systematically characterized the binding of T-ag to ssDNA and to synthetic DNA forks, in the presence or absence of nucleotide cofactors. We further demonstrated that T-ag forms a double hexamer in type II complexes but that both type I and type II complexes contain only a single DNA fork. These differences in DNA binding affinities can explain why type II complexes have much higher helicase activity than do type I complexes

EXPERIMENTAL PROCEDURES

RESULTS

Longer duplex

DISCUSSION