Helicase motif Ia is involved in single-strand DNA-binding and helicase activities of the herpes simplex virus type 1 origin-binding protein, UL9.
UL9 is a multifunctional protein essential for herpes simplex virus type 1 (HSV-1) replication in vivo. UL9 is a member of the superfamily II helicases and exhibits helicase and origin-binding activities. It is thought that UL9 binds the origin of replication and unwinds it in the presence of ATP and the HSV-1 single-stranded DNA (ssDNA)-binding protein. We have previously characterized the biochemical properties of mutants in all helicase motifs except for motif Ia (B. Marintcheva and S. Weller, J. Biol. Chem. 276:6605-6615, 2001). Structural information for other superfamily I and II helicases indicates that motif Ia is involved in ssDNA binding. By analogy, we hypothesized that UL9 motif Ia is important for the ssDNA-binding function of the protein. On the basis of sequence conservation between several UL9 homologs within the Herpesviridae family and distant homology with helicases whose structures have been solved, we designed specific mutations in motif Ia and analyzed them genetically and biochemically. Mutant proteins with residues predicted to be involved in ssDNA binding (R112A and R113A/F115A) exhibited wild-type levels of intrinsic ATPase activity and moderate to severe defects in ssDNA-stimulated ATPase activity and ssDNA binding. The S110T mutation targets a residue not predicted to contact ssDNA directly. The mutant protein with this mutation exhibited wild-type levels of intrinsic ATPase activity and near wild-type levels of ssDNA-stimulated ATPase activity and ssDNA binding. All mutant proteins lack helicase activity but were able to dimerize and bind the HSV-1 origin of replication as well as wild-type UL9. Our results indicate that residues from motif Ia contribute to the ssDNA-binding and helicase activities of UL9 and are essential for viral growth. This work represents the successful application of an approach based on a combination of bioinformatics and structural information from related proteins to deduce valuable information about a protein of interest.
Marintcheva
Boriana
B
Department of Microbiology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA.
Weller
Sandra K
SK
D000251:Adenosine Triphosphatases / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002522:Chlorocebus aethiops; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D018259:Herpesvirus 1, Human / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D018741:Replication Origin / Q000502:physiology*; D013329:Structure-Activity Relationship; D014162:Transfection; D014709:Vero Cells; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C064565:UL9 protein, Human herpesvirus 1; D000251:Adenosine Triphosphatases; D004265:DNA Helicases