A Biochemical Characterization of the Adeno-associated Virus Rep40 Helicase
Open Access
- 1 September 2003
- journal article
- Published by Elsevier BV
- Vol. 278 (36), 34011-34017
- https://doi.org/10.1074/jbc.m301537200
Abstract
The human adeno-associated virus (AAV) has generated much enthusiasm as a transfer vector for human gene therapy. Although clinical gene therapy trials have been initiated using AAV vectors, much remains to be learned regarding the basic mechanisms of virus replication, gene expression, and virion assembly. AAV encodes four nonstructural, or replication (Rep), proteins. The Rep78 and Rep68 proteins regulate viral DNA replication, chromosomal integration, and gene expression. The Rep52 and Rep40 proteins mediate virus assembly. To better understand Rep protein function, we have expressed the Rep40 protein in Escherichia coli and purified it to near homogeneity. Like the other Rep proteins, Rep40 possesses helicase and ATPase activity. ATP is the best substrate, and Mg2+ is the most efficient divalent metal ion for helicase activity. A Lys to His mutation in the purine nucleotide-binding site results in a protein that inhibits helicase activity in a dominant negative manner. Rep40 unwinds double-stranded DNA containing a 3′ single-stranded end, or blunt end, unlike the Rep68 and Rep52 enzymes, which have a strict requirement for DNA duplexes containing a 3′ single-stranded end. Values for KATP in the ATPase assay are 1.1 ± 0.2 mm and 1.2 ± 0.2 mm in the absence and presence, respectively, of single-stranded DNA. Values for Vmax are 220 ± 10 and 1,500 ± 90 nmol/min/mg in the absence and presence, respectively, of single-stranded DNA. These studies provide the first enzymatic characterization of the AAV Rep40 protein and elucidate important functional differences between the AAV helicases.Keywords
This publication has 9 references indexed in Scilit:
- DNA helicase-mediated packaging of adeno-associated virus type 2 genomes into preformed capsidsThe EMBO Journal, 2001
- Biochemical Characterization of the DNA Helicase Activity of the Escherichia coli RecQ HelicasePublished by Elsevier BV ,2001
- Ubiquitous Human Adeno-Associated Virus Type 2 Autonomously Replicates in Differentiating Keratinocytes of a Normal Skin ModelVirology, 2000
- Roles of Adeno-Associated Virus Rep Protein and Human Chromosome 19 in Site-Specific RecombinationJournal of Virology, 2000
- Helicase motifs: the engine that powers DNA unwindingMolecular Microbiology, 1999
- RecQ helicase, in concert with RecA and SSB proteins, initiates and disrupts DNA recombinationGenes & Development, 1998
- RecQ DNA Helicase of Escherichia coli: Characterization of the Helix-unwinding Activity with Emphasis on the Effect of Single-stranded DNA-binding ProteinJournal of Molecular Biology, 1993
- Calculation of protein extinction coefficients from amino acid sequence dataAnalytical Biochemistry, 1989
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976