Replacement of gly815 in helicase motif V alters the single-stranded DNA-dependent ATPase activity of the herpes simplex virus type 1 helicase-primase.
Herpes simplex virus type 1 encodes a helicase-primase complex composed of the products of the UL5, UL52, and UL8 genes. A subcomplex consisting of the UL5 and UL52 proteins purified from insect cells also displays ATPase, helicase, and primase activities. UL5 contains six motifs conserved in superfamily I of known and/or putative helicase proteins. Consistent with the ability to hydrolyze ATP, motifs I and II resemble a nucleotide binding site. Although the role of the other four motifs is not known, single amino acid substitutions created in conserved residues in all six motifs abolish the ability of UL5 to support viral DNA replication in vivo (Zhu, L., and Weller, S. K. (1992) J. Virol. 66, 469-479). In one such mutation, a highly conserved glycine in motif V (Gly815) is replaced with an alanine. Although the UL5(G815A) protein does not support viral DNA replication in vivo, the purified UL5(G815A).52 subcomplex retains primase and helicase activities and supports strand displacement DNA synthesis on a preformed replication fork in the presence of the other HSV-1 replication proteins. The major difference between the wild-type and variant protein is that the UL5(G815A).52 subcomplex displays an increased Km for single-stranded DNA and decreased Kcat for single-stranded DNA-dependent ATPase activity. Several hypotheses for the role of motif V in the function of the UL5 helicase in HSV-1 DNA replication are considered. This is the first report of a biochemical analysis of a motif V variant in any member of helicase superfamily I.
Graves-Woodward
K L
KL
Department of Microbiology, The University of Connecticut Health Center, Farmington, Connecticut 06030-3205, USA.
Weller
S K
SK
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D017124:Conserved Sequence; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019915:DNA Primase; D004277:DNA, Single-Stranded / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics*; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D018411:Spodoptera; D014764:Viral Proteins
D004277:DNA, Single-Stranded; D011994:Recombinant Proteins; D014764:Viral Proteins; D019915:DNA Primase; C062021:helicase-primase, Human herpesvirus 1; D000251:Adenosine Triphosphatases; C037693:single stranded DNA dependent ATPase; D004265:DNA Helicases