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J. Virol. doi:10.1128/JVI.00384-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A Quantitative Analysis of the Binding of Simian Virus 40 Large T Antigen to DNA

Laboratory of Molecular Virology, Institut de Recherches Cliniques de Montréal, 110 Pine Avenue West, Montreal, Quebec, Canada, H2W 1R7 and Department of Biochemistry, University of Montreal, Montreal, Quebec, Canada; Department of Biochemistry, Tufts University School of Medicine, Boston, Massachussetts 02111

^* To whom correspondence should be addressed. Email: jacques.archambault{at}ircm.qc.ca.

	Abstract

SV40 Large T antigen (T-ag) is a multifunctional protein that successively binds to 5'-GAGGC-3' sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork and interacts with host DNA replication factors to promote replication of the SV40 genome. The transition of T-ag from a sequence specific binding protein to a non specific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for non specific double- and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5'-GAGGC-3' sequence and of the residue downstream of it which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with a 10-fold higher affinity than to non specific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.

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