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Journal of Virology, June 2008, p. 6017-6023, Vol. 82, No. 12
0022-538X/08/$08.00+0     doi:10.1128/JVI.00387-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Systematic Study of the Functions for the Residues around the Nucleotide Pocket in Simian Virus 40 AAA+ Hexameric Helicase

William B. Greenleaf,^1, Jingping Shen,^2, Dahai Gai,¹ and Xiaojiang S. Chen^1*

Department of Molecular and Computational Biology and Department of Biochemistry and Molecular Genetics, University of Southern California, Los Angeles, California 90089-1340,¹ University of Colorado at Denver and Health Sciences Center, Aurora, Colorado 80045²

Received 22 February 2008/ Accepted 27 March 2008

The high-resolution structural data for simian virus 40 large-T-antigen helicase revealed a set of nine residues bound to ATP/ADP directly or indirectly. The functional role of each of these residues in ATP hydrolysis and also the helicase function of this AAA+ (ATPases associated with various cellular activities) molecular motor are unclear. Here, we report our mutational analysis of each of these residues to examine their functionality in oligomerization, DNA binding, ATP hydrolysis, and double-stranded DNA (dsDNA) unwinding. All mutants were capable of oligomerization in the presence of ATP and could bind single-stranded DNA and dsDNA. ATP hydrolysis was substantially reduced for proteins with mutations of residues making direct contact with the -phosphate of ATP or the apical water molecule. A potentially noncanonical "arginine finger" residue, K418, is critical for ATP hydrolysis and helicase function, suggesting a new type of arginine finger role by a lysine in the stabilization of the transition state during ATP hydrolysis. Interestingly, our mutational data suggest that the positive- and negative-charge interactions in the uniquely observed residue pairs, R498/D499 and R540/D502, in large-T-antigen helicase are critically involved in the transfer of energy of ATP binding/hydrolysis to DNA unwinding.

Published ahead of print on 9 April 2008.

These authors contributed equally to this work.