Journal
MOLECULAR AND CELLULAR BIOLOGY
Volume 22, Issue 18, Pages 6592-6604Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/MCB.22.18.6592-6604.2002
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Funding
- NCI NIH HHS [CA83679, R01 CA083679] Funding Source: Medline
- NIGMS NIH HHS [GM 31819, R01 GM031819] Funding Source: Medline
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Human papillomavirus (HPV) DNA replication requires the viral origin recognition protein E2 and the presumptive viral replicative helicase El. We now report for the first time efficient DNA unwinding by a purified HPV El 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 El 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 El binding to the origin and promote dihexameric El 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 El 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.
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