A Ring-opening Mechanism for DNA Binding in the Central Channel of the T7 Helicase-primase Protein

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 2000 Jul 6

PMID 10880454

Citations 34

Authors

P Ahnert

K M Picha

S S Patel

Affiliations

Soon will be listed here.

Abstract

We have investigated the mechanism of binding single-stranded DNA (ssDNA) into the central channel of the ring-shaped T7 gp4A' helicase-primase hexamer. Presteady-state kinetic studies show a facilitated five-step mechanism and provide understanding of how a ring-shaped helicase can be loaded on the DNA during the initiation of replication. The effect of a primase recognition sequence on the observed kinetics suggests that binding to the helicase DNA-binding site is facilitated by transient binding to the primase DNA-binding site, which is proposed to be a loading site. The proposed model involves the fast initial binding of the DNA to the primase site on the outside of the helicase ring, a fast conformational change, a ring-opening step, migration of the DNA into the central channel of the helicase ring, and ring closure. Although an intermediate protein-DNA complex is kinetically stable, only the last species in the five-step mechanism is poised to function as a helicase at the unwinding junction.

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References

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

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

Bujalowski W, Jezewska M . Kinetic mechanism of the single-stranded DNA recognition by Escherichia coli replicative helicase DnaB protein. Application of the matrix projection operator technique to analyze stopped-flow kinetics. J Mol Biol. 2000; 295(4):831-52. DOI: 10.1006/jmbi.1999.3378. View

Picha K, Ahnert P, Patel S . DNA binding in the central channel of bacteriophage T7 helicase-primase is a multistep process. Nucleotide hydrolysis is not required. Biochemistry. 2000; 39(21):6401-9. DOI: 10.1021/bi992857i. View

Patel S, Picha K . Structure and function of hexameric helicases. Annu Rev Biochem. 2000; 69:651-97. DOI: 10.1146/annurev.biochem.69.1.651. View

Cleland W . Partition analysis and the concept of net rate constants as tools in enzyme kinetics. Biochemistry. 1975; 14(14):3220-4. DOI: 10.1021/bi00685a029. View

Scherzinger E, LANKA E, Hillenbrand G . Role of bacteriophage T7 DNA primase in the initiation of DNA strand synthesis. Nucleic Acids Res. 1977; 4(12):4151-63. PMC: 343231. DOI: 10.1093/nar/4.12.4151. 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

Hingorani M, Patel S . Interactions of bacteriophage T7 DNA primase/helicase protein with single-stranded and double-stranded DNAs. Biochemistry. 1993; 32(46):12478-87. DOI: 10.1021/bi00097a028. View

10.

Patel S, Hingorani M, Ng W . The K318A mutant of bacteriophage T7 DNA primase-helicase protein is deficient in helicase but not primase activity and inhibits primase-helicase protein wild-type activities by heterooligomer formation. Biochemistry. 1994; 33(25):7857-68. DOI: 10.1021/bi00191a013. View

11.

Tarumi K, Yonesaki T . Functional interactions of gene 32, 41, and 59 proteins of bacteriophage T4. J Biol Chem. 1995; 270(6):2614-9. DOI: 10.1074/jbc.270.6.2614. View

12.

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

13.

Patel S, Hingorani M . Nucleotide binding studies of bacteriophage T7 DNA helicase-primase protein. Biophys J. 1995; 68(4 Suppl):186S-189S; discussion 189S-190S. PMC: 1281911. View

14.

Bujalowski W, Jezewska M . Interactions of Escherichia coli primary replicative helicase DnaB protein with single-stranded DNA. The nucleic acid does not wrap around the protein hexamer. Biochemistry. 1995; 34(27):8513-9. DOI: 10.1021/bi00027a001. View

15.

Hingorani M, Patel S . Cooperative interactions of nucleotide ligands are linked to oligomerization and DNA binding in bacteriophage T7 gene 4 helicases. Biochemistry. 1996; 35(7):2218-28. DOI: 10.1021/bi9521497. View

16.

Yu X, Hingorani M, Patel S, Egelman E . DNA is bound within the central hole to one or two of the six subunits of the T7 DNA helicase. Nat Struct Biol. 1996; 3(9):740-3. DOI: 10.1038/nsb0996-740. View

17.

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

18.

DONOVAN S, Harwood J, Drury L, Diffley J . Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. Proc Natl Acad Sci U S A. 1997; 94(11):5611-6. PMC: 20826. DOI: 10.1073/pnas.94.11.5611. View

19.

BIRD L, Hakansson K, Pan H, Wigley D . Characterization and crystallization of the helicase domain of bacteriophage T7 gene 4 protein. Nucleic Acids Res. 1997; 25(13):2620-6. PMC: 146783. DOI: 10.1093/nar/25.13.2620. View

20.

Tanaka T, Knapp D, Nasmyth K . Loading of an Mcm protein onto DNA replication origins is regulated by Cdc6p and CDKs. Cell. 1997; 90(4):649-60. DOI: 10.1016/s0092-8674(00)80526-7. View