Binding of the baculovirus very late expression factor 1 (VLF-1) to different DNA structures

VLF-1 was expressed in a recombinant baculovirus as a fusion with both HA and HIS6 tags and its binding activity to different DNA structures was tested. No binding was evident to single and double strand structures, very low binding was observed to Y-forks, more binding was observed to three-way junctions, whereas cruciform structures showed high levels of binding. VLF-1 binding was affected by divalent cations; optimal binding to three-way junctions and cruciforms was 2 and 0 mM MgCl2, respectively. Homologous region (hr) sequences was also examined including oligomers designed to expose the hr palindrome as a hairpin, linear double strand, or H-shaped structure. Efficient binding was observed to the hairpin and H-shaped structure. No topoisomerase or endonuclease activity was detected. Sedimentation analysis indicated that *VLF-1 is present as a monomer.An HA- and HIS-tagged version of AcMNPV VLF-1 showed structure-dependent binding to DNA substrates with the highest binding affinity to cruciform DNA. These results are consistent with the involvement of VLF-1 in the processing of branched DNA molecules at the late stages of viral genome replication. We were unable to detect enzymatic activity associated with these complexes.Baculoviruses contain double-stranded, circular, covalently closed DNA genomes of 100–180 kb [1,2]. A distinctive feature of their genomes is the presence of homologous regions (hrs) located at a number of positions. In the best characterized baculovirus, Autographa californica multiple capsid nucleopolyhedrovirus (AcMNPV), the hr repeat units contain about 70-bp with an imperfect 30-bp palindrome located near the center, and are repeated two to eight times at each of eight locations around the genome. Hrs have been implicated both as transcriptional enhancers and origins of DNA replication for a number of baculoviruses [3-9]. Hr-containing plasmid DNA transfected into AcMNPV-infected cells appears to replicate as high molecular weight concatem