The herpes simplex virus type 1 origin-binding protein carries out origin specific DNA unwinding and forms stem-loop structures.
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.
Makhov
A M
AM
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill 27599-7295, USA.
Boehmer
P E
PE
Lehman
I R
IR
Griffith
J D
JD
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D003432:Cross-Linking Reagents; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D010777:Photochemistry; D011485:Protein Binding; D018741:Replication Origin; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
D003432:Cross-Linking Reagents; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C064565:UL9 protein, Human herpesvirus 1; D000255:Adenosine Triphosphate; D004265:DNA Helicases