Coupled ATP and DNA binding of adeno-associated virus Rep40 helicase.
Adeno-associated virus 2 Rep40 helicase is involved in packaging single-stranded genomic DNA 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 indicates that 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 ATPgammaS; ADP and AMPPCP did not promote binding. The cooperativity and the nucleotide requirement were also demonstrated by surface plasmon resonance. Although the Rep40 behaves as a monomer in solution, it binds to DNA as an oligomer. The requirement of a nucleotide for DNA binding and the stimulation of ATPase activity by DNA indicate that the two processes are linked. Glutaraldehyde cross-linking generated a species that migrates as a trimer on sodium dodecyl sulfate (SDS) gel electrophoresis; ATPS promoted the formation of this species and higher order oligomers. The predominant cross-linked species was a trimer in the absence of ATPgammaS, regardless of whether duplex or single-stranded DNA was present. In the presence of duplex or single-stranded DNA and ATPgammaS, glutaraldehyde cross-linking generated a species that behaved as a dimer on SDS gel elctrophoresis. Sucrose-gradient velocity sedimentation of Rep40 gave an S20,w of 3 in the absence of ligands or in the presence of a 26 bp duplex DNA. The S20,w was 3.5 in the presence of ATPgammaS and 7 and 7.6 in the presence of DNA and ATPgammaS.
Dignam
Susan S
SS
Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, 3035 Arlington Avenue, Toledo, Ohio 43614-5804, USA.
Collaco
Roy F
RF
Bieszczad
Jacob
J
Needham
Patrick
P
Trempe
James P
JP
Dignam
John David
JD
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D003432:Cross-Linking Reagents; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics; D000229:Dependovirus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D019281:Dimerization; D004789:Enzyme Activation; D005454:Fluorescence Polarization; D005976:Glutaral; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009994:Osmolar Concentration; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly
D003432:Cross-Linking Reagents; D004279:DNA, Viral; D011994:Recombinant Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004265:DNA Helicases; C477740:Rep40 protein, adeno-associated virus; D005976:Glutaral