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Molecular and Cellular Biology, September 2002, p. 6592-6604, Vol. 22, No. 18
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.18.6592-6604.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Chaperone Proteins Abrogate Inhibition of the Human Papillomavirus (HPV) E1 Replicative Helicase by the HPV E2 Protein

Biing Yuan Lin,1 Alexander M. Makhov,2 Jack D. Griffith,2 Thomas R. Broker,1 and Louise T. Chow1*

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005,1 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-72952

Received 27 March 2002/ Returned for modification 10 May 2002/ Accepted 24 June 2002

Human papillomavirus (HPV) DNA replication requires the viral origin recognition protein E2 and the presumptive viral replicative helicase E1. We now report for the first time efficient DNA unwinding by a purified HPV E1 protein. Unwinding depends on a supercoiled DNA substrate, topoisomerase I, single-stranded-DNA-binding protein, and ATP, but not an origin. Electron microscopy revealed completely unwound molecules. Intermediates contained two single-stranded loops emanating from a single protein complex, suggesting a bidirectional E1 helicase which translocated the flanking DNA in an inward direction. We showed that E2 protein partially inhibited DNA unwinding and that Hsp70 or Hsp40, which we reported previously to stimulate HPV-11 E1 binding to the origin and promote dihexameric E1 formation, apparently displaced E2 and abolished inhibition. Neither E2 nor chaperone proteins were detected in unwinding complexes. These results suggest that chaperones play important roles in the assembly and activation of a replicative helicase in higher eukaryotes. An E1 mutation in the ATP binding site caused deficient binding and unwinding of origin DNA, indicating the importance of ATP binding in efficient helicase assembly on the origin.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 1918 University Blvd, MCLM 510, Birmingham, AL 35294-0005. Phone: (205) 975-8300. Fax: (205) 975-6075. E-mail: LTChow{at}uab.edu.

Molecular and Cellular Biology, September 2002, p. 6592-6604, Vol. 22, No. 18
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.18.6592-6604.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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