Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus.
DNA-binding protein (DBP) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was expressed as an N-terminal His(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.
Mikhailov
Victor S
VS
Department of Microbiology, Oregon State University, Corvallis, OR 97331-3804, USA. Victor.Mikhailov@orst.edu
Vanarsdall
Adam L
AL
Rohrmann
George F
GF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003432:Cross-Linking Reagents; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths / Q000821:virology*; D017924:Nucleopolyhedroviruses / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014357:Trypsin; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D003432:Cross-Linking Reagents; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D014764:Viral Proteins; D014357:Trypsin