Mutational analysis of the adeno-associated virus type 2 Rep68 protein helicase motifs.
AUTOR(ES)
Walker, S L
RESUMO
The adeno-associated virus type 2 (AAV) Rep78 and Rep68 proteins are required for viral replication. These proteins are encoded by unspliced and spliced transcripts, respectively, from the p5 promoter of AAV and therefore have overlapping amino acid sequences. The Rep78 and Rep68 proteins share a variety of activities including endonuclease, helicase, and ATPase activities and the ability to bind AAV hairpin DNA. The part of the amino acid sequence which is identical in Rep78 and Rep68 contains consensus helicase motifs that are conserved among the parvovirus replication proteins. In the present study, we mutated highly conserved amino acids within these helicase motifs. The mutant proteins were synthesized as maltose binding protein-Rep68 fusions in Escherichia coli cells and affinity purified on amylose resin. The fusion proteins were assayed in vitro, and their activities were directly compared to those of the fusion protein MBP-Rep68 delta, which contains most of the amino acid sequences common to Rep78 and Rep68 and was demonstrated previously to have all of the in vitro activities of wild-type Rep78 and Rep68. Our analysis showed that almost all mutations in the putative helicase motifs severely reduced or abolished helicase activity in vitro. Most mutants also had ATPase activity less than one-eighth of the wild-type levels and lacked endonuclease activity.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=191985Documentos Relacionados
- Mutational Analysis of Adeno-Associated Virus Type 2 Rep68 Protein Endonuclease Activity on Partially Single-Stranded Substrates
- Biochemical Characterization of Adeno-Associated Virus Rep68 DNA Helicase and ATPase Activities
- Mutational analysis of the adeno-associated virus Rep68 protein: identification of critical residues necessary for site-specific endonuclease activity.
- Analysis of the Effects of Charge Cluster Mutations in Adeno-Associated Virus Rep68 Protein In Vitro
- The Adeno-Associated Virus Type 2 Regulatory Proteins Rep78 and Rep68 Interact with the Transcriptional Coactivator PC4