THE HERPES-SIMPLEX VIRUS TYPE-1 ORIGIN-BINDING PROTEIN CARRIES OUT ORIGIN SPECIFIC DNA UNWINDING AND FORMS UNWOUND STEM-LOOP STRUCTURES

The UL9 protein of herpes simplex virus type 1 (HSV-1) binds specifica lly to the HSV-1 ori(S) and ori(L) origins of replication, and is a DN A helicase and DNA-dependent NTPase. In this study electron microscopy was used to investigate the binding of UL9 protein to DNA fragments c ontaining ori(S). In the absence of ATP, UL9 protein was observed to b ind specifically to ori(S) as a dimer or pair of dimers, which bent th e DNA by 35 degrees +/- 15 degrees and 86 degrees +/- 38 degrees respe ctively, and the DNA was deduced to make a straight line path through the protein complex. In the presence of 4 mM ATP, binding at ori(S) wa s enhanced 2-fold, DNA loops or stem-loops were extruded from the UL9 protein complex at ori(S), and the DNA in them frequently appeared hig hly condensed into a tight rod. The stem-loops contained from a few hu ndred to over one thousand base pairs of DNA and in most, ori(S) was l ocated at their apex, although in some, ori(S) was at a border. The DN A in the stem-loops could be stabilized by photocrosslinking, and when Escherichia coli SSB protein was added to the incubations, it bound t he stem-loops strongly. Thus the DNA strands in the stem-loops exist i n a partially paired, partially single-stranded state presumably makin g them available for ICP8 binding in vivo. These observations provide direct evidence for an origin specific unwinding by the HSV-1 UL9 prot ein and for the formation of a relatively stable four-stranded DNA in this process.