The herpes simplex virus type 1 origin-binding protein carries out origin specific DNA unwinding and forms stem-loop structures.

Abstract

The UL9 protein of herpes simplex virus type 1 (HSV‐1) binds specifically to the HSV‐1 oriS and oriL origins of replication, and is a DNA helicase and DNA‐dependent NTPase. In this study electron microscopy was used to investigate the binding of UL9 protein to DNA fragments containing oriS. In the absence of ATP, UL9 protein was observed to bind specifically to oriS as a dimer or pair of dimers, which bent the DNA by 35 degrees +/− 15 degrees and 86 degrees +/− 38 degrees respectively, and the DNA was deduced to make a straight line path through the protein complex. In the presence of 4 mM ATP, binding at oriS was enhanced 2‐fold, DNA loops or stem‐loops were extruded from the UL9 protein complex at oriS, and the DNA in them frequently appeared highly condensed into a tight rod. The stem‐loops contained from a few hundred to over one thousand base pairs of DNA and in most, oriS was located at their apex, although in some, oriS was at a border. The DNA in the stem‐loops could be stabilized by photocrosslinking, and when Escherichia coli SSB protein was added to the incubations, it bound the stem‐loops strongly. Thus the DNA strands in the stem‐loops exist in a partially paired, partially single‐stranded state presumably making them available for ICP8 binding in vivo. These observations provide direct evidence for an origin specific unwinding by the HSV‐1 UL9 protein and for the formation of a relatively stable four‐stranded DNA in this process.

Origin-specific unwinding of herpes simplex virus 1 DNA by the viral UL9 and ICP8 proteins: Visualization of a specific preunwinding complex

“…Unwinding was optimal near 37°C and was not observed if the A͞T spacer was replaced with a base-paired G͞C-rich spacer. Furthermore, in our earlier EM study, we observed that the assembly of a pair of UL9 dimers at oriS bent the DNA by at least 90° (29), an event that would clearly be facilitated by melting of the A͞T-rich spacer. Although it is not likely that HSV-1 DNA globally is under significant superhelical strain in vivo as it was in our in vitro studies, transcription through oriS could induce local negative superhelicity behind the moving transcription complex (44).…”

“…UL9 protein is a homodimer in solution, and several studies have shown that two dimers are bound to oriS containing all three binding boxes (9,10). Electron microscopy (EM) studies from this laboratory revealed a particle with a mass consistent with a double dimer assembled at oriS (29).…”

“…In our EM studies, using linear plasmid DNAs containing the minimal oriS segment, a double dimer was visualized bound at oriS, which bent the DNA Ϸ90° (29). After addition of ATP, stem-loop structures encompassing many hundreds of nucleotides were extruded from the UL9 complex, and the amount of DNA in the stem-loops increased with incubation time.…”

An initial ATP-independent step in the unwinding of a herpes simplex virus type I origin of replication by a complex of the viral origin-binding protein and single-strand DNA-binding protein

“…The resulting UL9 protein-ICP8 complex, presumably using the energy of binding, can then completely unwind the A ϩ T-rich sequence in an ATP-independent reaction. As observed previously, a specific UL9 protein-ICP8 complex is required (2-4); the E. coli SSB, although capable of binding to the A ϩ T-rich sequence (10), is unable to promote its unwinding, presumably because of its inability to interact with the UL9 protein. In the presence of an energy source, i.e., ATP, the UL9 protein-ICP8 complex can promote the complete unwinding of Ori s to permit access of the replication machinery to the origin sequences.…”

Herpes Virus Replication

“…This process has been demonstrated on a number of oriS containing substrates where unwinding generates long single-stranded stem-loop structures (58,59). This process may proceed via a conformational DNA intermediate, oriS*, that is formed by intrastrand hairpin formation at the origin (60).…”