Herpes simplex virus-1 DNA primase: a remarkably inaccurate yet selective polymerase.
Herpes simplex virus-1 primase misincorporates the natural NTPs at frequencies of around one error per 30 NTPs polymerized, making it one of the least accurate polymerases known. We used a series of nucleotide analogues to further test the hypothesis that primase requires Watson-Crick hydrogen bond formation to efficiently polymerize a NTP. Primase could not generate base pairs containing a complete set of hydrogen bonds in an altered arrangement (isoguanine.isocytosine) and did not efficiently polymerize dNTPs completely incapable of forming Watson-Crick hydrogen bonds opposite templating bases incapable of forming Watson-Crick hydrogen bonds. Similarly, primase did not incorporate most NTPs containing hydrophobic bases incapable of Watson-Crick hydrogen bonding opposite natural template bases. However, 2-pyridone NTP and 4-methyl-2-pyridone NTP provided striking exceptions to this rule. The effects of removing single Watson-Crick hydrogen bonding groups from either the NTP or templating bases varied from almost no effect to completely blocking polymerization depending both on the parental base pair (G.C vs A.T/U) and which base pair of the growing primer (second, third, or fourth) was examined. Thus, primase does not absolutely need to form Watson-Crick hydrogen bonds to efficiently polymerize a NTP. Additionally, we found that herpes primase can misincorporate nucleotides both by misreading the template and by a primer-template slippage mechanism. The mechanistic and biological implications of these results are discussed.
Urban
Milan
M
Department of Chemistry and Biochemistry, University of Colorado, UCB 215, Boulder, Colorado 80309, USA.
Joubert
Nicolas
N
Hocek
Michal
M
Alexander
Richard E
RE
Kuchta
Robert D
RD
D020029:Base Pairing; D055162:Biocatalysis; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D006860:Hydrogen Bonding; D015394:Molecular Structure; D009838:Oligodeoxyribonucleotides; D012313:RNA / Q000096:biosynthesis; D012265:Ribonucleotides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
D003854:Deoxyribonucleotides; D009838:Oligodeoxyribonucleotides; C034477:RNA primers; D012265:Ribonucleotides; D014764:Viral Proteins; D012313:RNA; D019915:DNA Primase; C062021:helicase-primase, Human herpesvirus 1; D004265:DNA Helicases