DNA binding and aggregation properties of the vaccinia virus I3L gene product

The vaccinia virus I3L gene encodes a single-stranded DNA-binding protein w hich may play a role in viral replication and genetic recombination. We hav e purified native and recombinant forms of gpI3L and characterized both the DNA-binding reaction and the structural properties of DNA-protein complexe s. The purified proteins displayed anomalous electrophoretic properties in the presence of sodium dodecyl sulfate, behaving as if they were 4-kDa larg er than the true mass. Agarose gel shift analysis was used to monitor the f ormation of complexes composed of single-stranded DNA plus gpI3L protein, T hese experiments detected two different DNA binding modes whose formation w as dependent upon the protein density. The transition between the two bindi ng 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 aggre gates from which double-stranded DNA was excluded. Electron microscopy show ed that, in the absence of magnesium and at low protein densities, gpI3L fo rms 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 E scherichia coli single-stranded DNA protein. Moreover, the capacity to aggr egate DNA may provide an environment in which hybrid DNA formation could oc cur during DNA replication.