Chaperone proteins abrogate inhibition of the human papillomavirus (HPV) E1 replicative helicase by the HPV E2 protein.
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.
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 35294-0005, USA.
Biing Yuan
BY
Lin
Alexander M
AM
Makhov
Jack D
JD
Griffith
Thomas R
TR
Broker
Louise T
LT
Chow
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002474:Cell-Free System; D002738:Chloroquine / Q000494:pharmacology; D004247:DNA / Q000648:ultrastructure; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D007150:Immunohistochemistry; D007313:Insecta; D008854:Microscopy, Electron; D008954:Models, Biological; D009154:Mutation; D009690:Nucleic Acid Conformation; D027383:Papillomaviridae / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; C084304:E1 protein, Human papillomavirus type 11; D014764:Viral Proteins; D002738:Chloroquine; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D004264:DNA Topoisomerases, Type I