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Journal of Virology, May 2003, p. 5512-5518, Vol. 77, No. 9
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.9.5512-5518.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of the DNA-Binding Properties of the Origin-Binding Domain of Simian Virus 40 Large T Antigen by Fluorescence Anisotropy

S. Titolo, E. Welchner, P. W. White, and J. Archambault*

Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd., Laval, Canada H7S 2G5

Received 26 September 2002/ Accepted 3 February 2003

The affinity of the origin-binding domain (OBD) of simian virus 40 large T antigen for its cognate origin was measured at equilibrium using a DNA binding assay based on fluorescence anisotropy. At a near-physiological concentration of salt, the affinities of the OBD for site II and the core origin were 31 and 50 nM, respectively. Binding to any of the four 5'-GAGGC-3' binding sites in site II was only slightly weaker, between 57 and 150 nM. Although the OBD was shown previously to assemble as a dimer on two binding sites spaced by 7 bp, we found that increasing the distance between both binding sites by 1 to 3 bp had little effect on affinity. Similar results were obtained for full-length T antigen in absence of nucleotide. Addition of ADP-Mg, which promotes hexamerization of T antigen, greatly increased the affinity of full-length T antigen for the core origin and for nonspecific DNA. The implications of these findings for the assembly of T antigen at the origin and its transition to a non-specific DNA helicase are discussed.

* Corresponding author. Mailing address: Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd., 2100 Cunard St., Laval, Canada H7S 2G5. Phone: (450) 682-4640. Fax: (450) 682-4642. E-mail: jarchambault{at}lav.boehringer-ingelheim.com.

Journal of Virology, May 2003, p. 5512-5518, Vol. 77, No. 9
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.9.5512-5518.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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