Coupled ATP and DNA Binding of Adeno-Associated Virus Rep40 Helicase

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• 作者：Susan S. Dignam ; Roy F. Collaco ; Jacob Bieszczad ; Patrick Needham ; James P. Trempe ; John David Dignam
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：January 16, 2007
• 年：2007
• 卷：46
• 期：2
• 页码：568 - 576
• 全文大小：257K
• 年卷期：v.46,no.2(January 16, 2007)
• ISSN：1520-4995

文摘

Adeno-associated virus 2 Rep40 helicase is involved in packaging single-stranded genomicDNA into virions. ATPase activity was stimulated 5-10-fold by DNA, depending upon assay conditions.The concentration dependence of Rep40 ATPase activity in the absence and presence of DNA indicatesthat the monomer is inactive and that the active enzyme is at least a dimer. Binding to oligonucleotides,examined by fluorescence anisotropy, was positively cooperative and required ATP or ATPS; ADP andAMPPCP did not promote binding. The cooperativity and the nucleotide requirement were alsodemonstrated by surface plasmon resonance. Although the Rep40 behaves as a monomer in solution, itbinds to DNA as an oligomer. The requirement of a nucleotide for DNA binding and the stimulation ofATPase activity by DNA indicate that the two processes are linked. Glutaraldehyde cross-linking generateda species that migrates as a trimer on sodium dodecyl sulfate (SDS) gel electrophoresis; ATPS promotedthe formation of this species and higher order oligomers. The predominant cross-linked species was atrimer in the absence of ATPS, regardless of whether duplex or single-stranded DNA was present. Inthe presence of duplex or single-stranded DNA and ATPS, glutaraldehyde cross-linking generated aspecies that behaved as a dimer on SDS gel elctrophoresis. Sucrose-gradient velocity sedimentation ofRep40 gave an S_20,w of 3 in the absence of ligands or in the presence of a 26 bp duplex DNA. The S_20,wwas 3.5 in the presence of ATPS and 7 and 7.6 in the presence of DNA and ATPS.