Promiscuous Usage of Nucleotides by the DNA Helicase of Bacteriophage T7: Determinants of Nucleotide Specificity

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2009 Mar 20

PMID 19297330

Citations 10

Authors

Ajit K Satapathy

Donald J Crampton

Benjamin B Beauchamp

Charles C Richardson

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

Study of SV40 large T antigen nucleotide specificity for DNA unwinding.

Wang D, Alvarez-Cabrera A, Chen X Virol J. 2017; 14(1):79.

PMID: 28410592 PMC: 5391581. DOI: 10.1186/s12985-017-0733-5.

Discrete interactions between bacteriophage T7 primase-helicase and DNA polymerase drive the formation of a priming complex containing two copies of DNA polymerase.

Wallen J, Majka J, Ellenberger T Biochemistry. 2013; 52(23):4026-36.

PMID: 23675753 PMC: 3750080. DOI: 10.1021/bi400284j.

Choreography of bacteriophage T7 DNA replication.

Lee S, Richardson C Curr Opin Chem Biol. 2011; 15(5):580-6.

PMID: 21907611 PMC: 3195405. DOI: 10.1016/j.cbpa.2011.07.024.

Coupling dTTP hydrolysis with DNA unwinding by the DNA helicase of bacteriophage T7.

Satapathy A, Kulczyk A, Ghosh S, van Oijen A, Richardson C J Biol Chem. 2011; 286(39):34468-78.

PMID: 21840995 PMC: 3190778. DOI: 10.1074/jbc.M111.283796.

The mitochondrial DNA helicase TWINKLE can assemble on a closed circular template and support initiation of DNA synthesis.

Jemt E, Farge G, Backstrom S, Holmlund T, Gustafsson C, Falkenberg M Nucleic Acids Res. 2011; 39(21):9238-49.

PMID: 21840902 PMC: 3241658. DOI: 10.1093/nar/gkr653.

References

Matson S, Richardson C . Nucleotide-dependent binding of the gene 4 protein of bacteriophage T7 to single-stranded DNA. J Biol Chem. 1985; 260(4):2281-7. View

Tabor S, Richardson C . Template recognition sequence for RNA primer synthesis by gene 4 protein of bacteriophage T7. Proc Natl Acad Sci U S A. 1981; 78(1):205-9. PMC: 319020. DOI: 10.1073/pnas.78.1.205. View

Lee S, Richardson C . Essential lysine residues in the RNA polymerase domain of the gene 4 primase-helicase of bacteriophage T7. J Biol Chem. 2001; 276(52):49419-26. DOI: 10.1074/jbc.M108443200. View

Moreau M, McGeoch A, Lowe A, Itzhaki L, Bell S . ATPase site architecture and helicase mechanism of an archaeal MCM. Mol Cell. 2007; 28(2):304-14. DOI: 10.1016/j.molcel.2007.08.013. View

Lohman T, Bjornson K . Mechanisms of helicase-catalyzed DNA unwinding. Annu Rev Biochem. 1996; 65:169-214. DOI: 10.1146/annurev.bi.65.070196.001125. View

Crampton D, Guo S, Johnson D, Richardson C . The arginine finger of bacteriophage T7 gene 4 helicase: role in energy coupling. Proc Natl Acad Sci U S A. 2004; 101(13):4373-8. PMC: 384754. DOI: 10.1073/pnas.0400968101. View

Sawaya M, Guo S, Tabor S, Richardson C, Ellenberger T . Crystal structure of the helicase domain from the replicative helicase-primase of bacteriophage T7. Cell. 1999; 99(2):167-77. DOI: 10.1016/s0092-8674(00)81648-7. View

Mendelman L, Notarnicola S, Richardson C . Roles of bacteriophage T7 gene 4 proteins in providing primase and helicase functions in vivo. Proc Natl Acad Sci U S A. 1992; 89(22):10638-42. PMC: 50396. DOI: 10.1073/pnas.89.22.10638. View

Patel S, Rosenberg A, Studier F, Johnson K . Large scale purification and biochemical characterization of T7 primase/helicase proteins. Evidence for homodimer and heterodimer formation. J Biol Chem. 1992; 267(21):15013-21. View

10.

Lee S, Qimron U, Richardson C . Communication between subunits critical to DNA binding by hexameric helicase of bacteriophage T7. Proc Natl Acad Sci U S A. 2008; 105(26):8908-13. PMC: 2449338. DOI: 10.1073/pnas.0802732105. View

11.

Lechner R, Richardson C . A preformed, topologically stable replication fork. Characterization of leading strand DNA synthesis catalyzed by T7 DNA polymerase and T7 gene 4 protein. J Biol Chem. 1983; 258(18):11185-96. View

12.

Mendelman L, Notarnicola S, Richardson C . Evidence for distinct primase and helicase domains in the 63-kDa gene 4 protein of bacteriophage T7. Characterization of nucleotide binding site mutant. J Biol Chem. 1993; 268(36):27208-13. View

13.

Egelman E, Yu X, Wild R, Hingorani M, Patel S . Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases. Proc Natl Acad Sci U S A. 1995; 92(9):3869-73. PMC: 42063. DOI: 10.1073/pnas.92.9.3869. View

14.

Kim D, Narayan M, Patel S . T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA. J Mol Biol. 2002; 321(5):807-19. DOI: 10.1016/s0022-2836(02)00733-7. View

15.

Kolodner R, Richardson C . Replication of duplex DNA by bacteriophage T7 DNA polymerase and gene 4 protein is accompanied by hydrolysis of nucleoside 5'-triphosphates. Proc Natl Acad Sci U S A. 1977; 74(4):1525-9. PMC: 430822. DOI: 10.1073/pnas.74.4.1525. View

16.

Notarnicola S, Park K, Griffith J, Richardson C . A domain of the gene 4 helicase/primase of bacteriophage T7 required for the formation of an active hexamer. J Biol Chem. 1995; 270(34):20215-24. DOI: 10.1074/jbc.270.34.20215. View

17.

Venkatesan M, Silver L, Nossal N . Bacteriophage T4 gene 41 protein, required for the synthesis of RNA primers, is also a DNA helicase. J Biol Chem. 1982; 257(20):12426-34. View

18.

Matson S, Kaiser-Rogers K . DNA helicases. Annu Rev Biochem. 1990; 59:289-329. DOI: 10.1146/annurev.bi.59.070190.001445. View

19.

Frick D, Richardson C . DNA primases. Annu Rev Biochem. 2001; 70:39-80. DOI: 10.1146/annurev.biochem.70.1.39. View

20.

Subramanya H, BIRD L, Brannigan J, Wigley D . Crystal structure of a DExx box DNA helicase. Nature. 1996; 384(6607):379-83. DOI: 10.1038/384379a0. View