DNA binding and aggregation properties of the vaccinia virus I3L gene product.
The vaccinia virus I3L gene encodes a single-stranded DNA-binding protein which may play a role in viral replication and genetic recombination. We have purified native and recombinant forms of gpI3L and characterized both the DNA-binding reaction and the structural properties of DNA-protein complexes. The purified proteins displayed anomalous electrophoretic properties in the presence of sodium dodecyl sulfate, behaving as if they were 4-kDa larger than the true mass. Agarose gel shift analysis was used to monitor the formation of complexes composed of single-stranded DNA plus gpI3L protein. These experiments detected two different DNA binding modes whose formation was dependent upon the protein density. The transition between the two binding modes occurred at a nucleotide to protein ratio of about 31 nucleotides per gpI3L monomer. S1 nuclease protection assay revealed that at saturating protein densities, each gpI3L monomer occludes 9.5 +/- 2.5 nucleotides. In the presence of magnesium, gpI3L promoted the formation of large DNA aggregates from which double-stranded DNA was excluded. Electron microscopy showed that, in the absence of magnesium and at low protein densities, gpI3L forms beaded structures on DNA. At high protein density the complexes display a smoother and less compacted morphology. In the presence of magnesium the complexes contained long fibrous and tangled arrays. These results suggest that gpI3L can form octameric complexes on DNA much like those formed by Escherichia coli single-stranded DNA protein. Moreover, the capacity to aggregate DNA may provide an environment in which hybrid DNA formation could occur during DNA replication.
Department of Molecular Biology & Genetics, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
M
M
Tseng
N
N
Palaniyar
W
W
Zhang
D H
DH
Evans
D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008854:Microscopy, Electron; D008961:Models, Structural; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C120652:I3L protein, Vaccinia virus; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; D008274:Magnesium