Key role of template sequence for primer synthesis by the herpes simplex virus 1 helicase-primase.
We investigated the effects of ssDNA template sequence on both primer synthesis and NTP hydrolysis by herpes simplex virus 1 helicase-primase. Primer synthesis was found to be profoundly dependent upon template sequence. Although not absolutely required, an important sequence feature for significant production of longer primers (beyond four nucleotides in length) is a deoxyguanylate-pyrimidine-pyrimidine (3'-G-pyr-pyr-5') triplet in the template. The deoxyguanylate serves both to direct primase to initiate synthesis opposite the adjacent pyrimidine and to dramatically increase primer length. While primase synthesized significantly more long primers on those templates containing a G-pyr-pyr triplet, the enzyme still synthesized massive quantities of di- and trinucleotides on many templates containing this sequence. Varying the sequences around the G-pyr-pyr recognition sequence dramatically altered both the rate of primer synthesis and the fraction of primers longer than four nucleotides, indicating that primase must interact with both the G-pyr-pyr and flanking sequences in the template. In contrast to the large effects that varying the template sequence had on primase activity, ssDNA-dependent NTPase activity was essentially unaffected by changes in template sequence, including the presence or absence of the G-pyr-pyr trinucleotide. In addition to hydrolyzing NTPs the NTPase could also hydrolyze the 5'-terminal phosphate from newly synthesized primers.
Ramirez-Aguilar
Kathryn A
KA
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
Low-Nam
Nisha A
NA
Kuchta
Robert D
RD
D017766:Acid Anhydride Hydrolases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D010584:Bacteriophage phi X 174 / Q000201:enzymology; D001483:Base Sequence; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism; D019915:DNA Primase; D017931:DNA Primers / Q000096:biosynthesis* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry; D003848:Deoxyguanine Nucleotides / Q000737:chemistry; D015226:Dinucleoside Phosphates / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000502:physiology; D043583:Nucleoside-Triphosphatase; D010761:Phosphorus Radioisotopes / Q000737:chemistry; D019920:Photoaffinity Labels / Q000737:chemistry; D011742:Pyrimidine Nucleotides / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid; D013698:Templates, Genetic; D024363:Transcription Initiation Site; D014544:Uridine Triphosphate / Q000737:chemistry; D014764:Viral Proteins; D014779:Virus Replication
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D003848:Deoxyguanine Nucleotides; D015226:Dinucleoside Phosphates; D010761:Phosphorus Radioisotopes; D019920:Photoaffinity Labels; D011742:Pyrimidine Nucleotides; D014764:Viral Proteins; C077233:2'-deoxyguanylic acid; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D019915:DNA Primase; C062021:helicase-primase, Human herpesvirus 1; D017766:Acid Anhydride Hydrolases; D043583:Nucleoside-Triphosphatase; D004265:DNA Helicases; D014544:Uridine Triphosphate