Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus
Abstract
DNA-binding protein (DBP) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was expressed as an N-terminal His{sub 6}-tag fusion using a recombinant baculovirus and purified to near homogeneity. Purified DBP formed oligomers that were crosslinked by redox reagents resulting in predominantly protein dimers and tetramers. In gel retardation assays, DBP showed a high affinity for single-stranded oligonucleotides and was able to compete with another baculovirus SSB protein, LEF-3, for binding sites. DBP binding protected ssDNA against hydrolysis by a baculovirus alkaline nuclease AN/LEF-3 complex. Partial proteolysis by trypsin revealed a domain structure of DBP that is required for interaction with DNA and that can be disrupted by thermal treatment. Binding to ssDNA, but not to dsDNA, changed the pattern of proteolytic fragments of DBP indicating adjustments in protein structure upon interaction with ssDNA. DBP was capable of unwinding short DNA duplexes and also promoted the renaturation of long complementary strands of ssDNA into duplexes. The unwinding and renaturation activities of DBP, as well as the DNA binding activity, were sensitive to sulfhydryl reagents and were inhibited by oxidation of thiol groups with diamide or by alkylation with N-ethylmaleimide. A high affinity of DBP for ssDNA and its unwinding and renaturation activities confirmedmore »
- Authors:
-
- Department of Microbiology, Oregon State University, Corvallis, OR 97331-3804 (United States)
- Publication Date:
- OSTI Identifier:
- 21078013
- Resource Type:
- Journal Article
- Journal Name:
- Virology
- Additional Journal Information:
- Journal Volume: 370; Journal Issue: 2; Other Information: DOI: 10.1016/j.virol.2007.09.001; PII: S0042-6822(07)00588-0; Copyright (c) 2007 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0042-6822
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 60 APPLIED LIFE SCIENCES; AFFINITY; ALKYLATION; CROSS-LINKING; DIMERS; DOMAIN STRUCTURE; GELS; HEAT TREATMENTS; HYDROLYSIS; OLIGONUCLEOTIDES; OXIDATION; PROTEIN STRUCTURE; PROTEOLYSIS; THIOLS; TRYPSIN
Citation Formats
Mikhailov, Victor S, N. K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117808, Vanarsdall, Adam L, and Rohrmann, George F. Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus. United States: N. p., 2008.
Web. doi:10.1016/j.virol.2007.09.001.
Mikhailov, Victor S, N. K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117808, Vanarsdall, Adam L, & Rohrmann, George F. Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus. United States. https://doi.org/10.1016/j.virol.2007.09.001
Mikhailov, Victor S, N. K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117808, Vanarsdall, Adam L, and Rohrmann, George F. 2008.
"Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus". United States. https://doi.org/10.1016/j.virol.2007.09.001.
@article{osti_21078013,
title = {Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus},
author = {Mikhailov, Victor S and N. K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117808 and Vanarsdall, Adam L and Rohrmann, George F},
abstractNote = {DNA-binding protein (DBP) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was expressed as an N-terminal His{sub 6}-tag fusion using a recombinant baculovirus and purified to near homogeneity. Purified DBP formed oligomers that were crosslinked by redox reagents resulting in predominantly protein dimers and tetramers. In gel retardation assays, DBP showed a high affinity for single-stranded oligonucleotides and was able to compete with another baculovirus SSB protein, LEF-3, for binding sites. DBP binding protected ssDNA against hydrolysis by a baculovirus alkaline nuclease AN/LEF-3 complex. Partial proteolysis by trypsin revealed a domain structure of DBP that is required for interaction with DNA and that can be disrupted by thermal treatment. Binding to ssDNA, but not to dsDNA, changed the pattern of proteolytic fragments of DBP indicating adjustments in protein structure upon interaction with ssDNA. DBP was capable of unwinding short DNA duplexes and also promoted the renaturation of long complementary strands of ssDNA into duplexes. The unwinding and renaturation activities of DBP, as well as the DNA binding activity, were sensitive to sulfhydryl reagents and were inhibited by oxidation of thiol groups with diamide or by alkylation with N-ethylmaleimide. A high affinity of DBP for ssDNA and its unwinding and renaturation activities confirmed identification of DBP as a member of the SSB/recombinase family. These activities and a tight association with subnuclear structures suggests that DBP is a component of the virogenic stroma that is involved in the processing of replicative intermediates.},
doi = {10.1016/j.virol.2007.09.001},
url = {https://www.osti.gov/biblio/21078013},
journal = {Virology},
issn = {0042-6822},
number = 2,
volume = 370,
place = {United States},
year = {Sun Jan 20 00:00:00 EST 2008},
month = {Sun Jan 20 00:00:00 EST 2008}
}