REPLACEMENT OF GLY(815) 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 compose d of the products of the UL5, UL52, and UL8 genes, A subcomplex consis ting of the UL5 and UL52 proteins purified from insect cells also disp lays 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 resemb le a nucleotide binding site, Although the role of the other four moti fs 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 moti f V (Gly(815)) is replaced with an alanine, Although the UL5(G815A) pr otein does not support viral DNA replication in vivo, the purified UL5 (G815A). 52 subcomplex retains primase and helicase activities and sup ports strand displacement DNA synthesis on a preformed replication for k in the presence of the other HSV-1 replication proteins, The major d ifference between the wildtype and variant protein is that the UL5(G81 5A). 52 subcomplex displays an increased K-m for single-stranded DNA a nd decreased K-cat for single-stranded DNA-dependent ATPase activity, Several hypotheses for the role of motif V in the function of the UL5 helicase in HSV-I DNA replication are considered, This is the first re port of a biochemical analysis of a motif V variant in any member of h elicase superfamily I.