CHARACTERIZATION OF THE NUCLEOTIDE AND DNA COEFFECTOR BINDING-SITES OF THE HERPES-SIMPLEX VIRUS TYPE-1 (HSV-1) ENCODED HELICASE-PRIMASE COMPLEX AND UL9 ORIGIN-BINDING PROTEIN

Nucleotide and DNA coeffector substrate binding site characterisations were performed on two HSV-1 DNA helicases fulfilling different roles in DNA replication. Single ATP-binding sites were identified for helic ase-primase and UL9 protein (Km(ATP) 0.62 mM and 0.54 mM, respectively ). Analysis of structural requirements for DNA-dependent NTP hydrolysi s revealed comparatively stringent requirements for helicase-primase i n accommodating base-modified NTP analogs whereas the UL9 protein was much more permissive in this respect; neither enzyme was dependent on the ribose 2' or 3' hydroxyls for NTP hydrolysis. Both helicase-primas e and UL9 protein ATPase activities were inhibited by ADP or GDP; this effect was competitive rather than allosteric. The enhancement of ATP ase activity on a single stranded (ss) DNA substrate as opposed to dou ble stranded (ds) DNA was much more marked for helicase-primase than f or the UL9 protein (K(m(dsDNA))/K(m(ssDNA)) 60 and 9, respectively). T he triphosphates of the antiviral agents acyclovir and penciclovir wer e not effective substrates for either helicase-primase or UL9 protein. (C) 1994 Academic Press, Inc.