Promiscuous usage of nucleotides by the DNA helicase of bacteriophage T7: determinants of nucleotide specificity.
The multifunctional protein encoded by gene 4 of bacteriophage T7 (gp4) provides both helicase and primase activity at the replication fork. T7 DNA helicase preferentially utilizes dTTP to unwind duplex DNA in vitro but also hydrolyzes other nucleotides, some of which do not support helicase activity. Very little is known regarding the architecture of the nucleotide binding site in determining nucleotide specificity. Crystal structures of the T7 helicase domain with bound dATP or dTTP identified Arg-363 and Arg-504 as potential determinants of the specificity for dATP and dTTP. Arg-363 is in close proximity to the sugar of the bound dATP, whereas Arg-504 makes a hydrogen bridge with the base of bound dTTP. T7 helicase has a serine at position 319, whereas bacterial helicases that use rATP have a threonine in the comparable position. Therefore, in the present study we have examined the role of these residues (Arg-363, Arg-504, and Ser-319) in determining nucleotide specificity. Our results show that Arg-363 is responsible for dATP, dCTP, and dGTP hydrolysis, whereas Arg-504 and Ser-319 confer dTTP specificity. Helicase-R504A hydrolyzes dCTP far better than wild-type helicase, and the hydrolysis of dCTP fuels unwinding of DNA. Substitution of threonine for serine 319 reduces the rate of hydrolysis of dTTP without affecting the rate of dATP hydrolysis. We propose that different nucleotides bind to the nucleotide binding site of T7 helicase by an induced fit mechanism. We also present evidence that T7 helicase uses the energy derived from the hydrolysis of dATP in addition to dTTP for mediating DNA unwinding.
Satapathy
Ajit K
AK
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Crampton
Donald J
DJ
Beauchamp
Benjamin B
BB
Richardson
Charles C
CC
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D019943:Amino Acid Substitution; D001120:Arginine; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000254:growth & development; D018360:Crystallography, X-Ray; D003570:Cytidine Triphosphate / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D050505:Mutant Proteins / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism* / Q000494:pharmacology; D010957:Plasmids; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D013379:Substrate Specificity / Q000187:drug effects; D013942:Thymine Nucleotides / Q000494:pharmacology
D050505:Mutant Proteins; D009711:Nucleotides; D013942:Thymine Nucleotides; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D001120:Arginine; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases; C024157:thymidine 5'-triphosphate