Abstract:
:Helicases are enzymes that use energy from nucleoside triphosphate hydrolysis to unwind double-stranded (ds) DNA, a process vital to virtually every phase of DNA metabolism. Helicases have been classified as either 5'-to-3' or 3'-to-5' on the basis of their ability to unwind duplex DNA adjacent to either a 5' or 3' single-stranded (ss) DNA overhang. However, there has been debate as to whether this substrate preference is indicative of unidirectional translocation on ssDNA. We developed an assay that monitors the ability of a helicase to displace streptavidin from biotinylated oligonucleotides [Morris, P. D., and Raney, K. D. (1999) Biochemistry 38, 5164-5171]. Two helicases identified as having 5'-to-3' polarity displaced streptavidin from the 3'-end of biotinylated oligonucleotides but not from the 5'-end. We performed similar experiments using the 3'-to-5' helicases from the hepatitis C virus (NS3) and SV40 virus (SV40 T antigen). NS3 and SV40 T antigen were able to displace streptavidin from a 5'-biotinylated oligonucleotide but not from a 3'-biotinylated oligonucleotide. NS3 and SV40 T antigen enhanced the spontaneous rate of dissociation of streptavidin from biotin 340-fold and 1700-fold, respectively. The ssDNA binding protein, gp32, did not enhance dissociation of streptavidin from either end of an oligonucleotide. For NS3, the rate of displacement was faster from a 5'-biotinylated 60mer than from a 5'-biotinylated 30mer. The strong directional bias in streptavidin displacement activity exhibited by each helicase is consistent with a directional bias in translocation on ssDNA. The dependence of the reaction with NS3 on the oligonucleotide length suggests that multiple NS3 monomers are necessary for optimal activity.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Morris PD,Byrd AK,Tackett AJ,Cameron CE,Tanega P,Ott R,Fanning E,Raney KDdoi
10.1021/bi012058bsubject
Has Abstractpub_date
2002-02-19 00:00:00pages
2372-8issue
7eissn
0006-2960issn
1520-4995pii
bi012058bjournal_volume
41pub_type
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