Linear Diffusion of the Restriction Endonuclease EcoRV on DNA is Essential for the in Vivo Function of the Enzyme

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 1996 Sep 16

PMID 8890184

Citations 29

Authors

A Jeltsch

C Wenz

F Stahl

A Pingoud

Affiliations

Soon will be listed here.

Abstract

Linear diffusion along DNA is a mechanism of enhancing the association rates of proteins to their specific recognition sites on DNA. It has been demonstrated for several proteins in vitro, but to date in no case in vivo. Here we show that the restriction endonuclease EcoRV slides along the DNA, scanning approximately 1000 bp in one binding event. This process is critically dependent on contacts between amino acid residues of the protein and the backbone of the DNA. The disruption of single hydrogen bonds and, in particular, the alteration of electrostatic interactions between amino acid side chains of the protein and phosphate groups of the DNA interfere with or abolish effective sliding. The efficiency of linear diffusion is dependent on salt concentration, having a maximum at 50 mM NaCl. These results suggest that a nonspecific and mobile binding mode capable of linear diffusion is dependent on a subtle balance of forces governing the interaction of the enzyme and the DNA. A strong correlation between the ability of EcoRV mutants to slide along the DNA in vitro and to protect Escherichia coli cells from phage infection demonstrates that linear diffusion occurs in vivo and is essential for effective phage restriction.

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References

Richter P, Eigen M . Diffusion controlled reaction rates in spheroidal geometry. Application to repressor--operator association and membrane bound enzymes. Biophys Chem. 1974; 2(3):255-63. DOI: 10.1016/0301-4622(74)80050-5. View

Thielking V, Alves J, Fliess A, Maass G, Pingoud A . Accuracy of the EcoRI restriction endonuclease: binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition sequences. Biochemistry. 1990; 29(19):4682-91. DOI: 10.1021/bi00471a024. View

Kim Y, Grable J, Love R, Greene P, Rosenberg J . Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing. Science. 1990; 249(4974):1307-9. DOI: 10.1126/science.2399465. View

Lesser D, Kurpiewski M . The energetic basis of specificity in the Eco RI endonuclease--DNA interaction. Science. 1990; 250(4982):776-86. DOI: 10.1126/science.2237428. View

Thielking V, Selent U, Kohler E, Wolfes H, Pieper U, Geiger R . Site-directed mutagenesis studies with EcoRV restriction endonuclease to identify regions involved in recognition and catalysis. Biochemistry. 1991; 30(26):6416-22. DOI: 10.1021/bi00240a011. View

Taylor J, Badcoe I, Clarke A, Halford S . EcoRV restriction endonuclease binds all DNA sequences with equal affinity. Biochemistry. 1991; 30(36):8743-53. DOI: 10.1021/bi00100a005. View

Aiken C, Fisher E, GUMPORT R . The specific binding, bending, and unwinding of DNA by RsrI endonuclease, an isoschizomer of EcoRI endonuclease. J Biol Chem. 1991; 266(28):19063-9. View

Thielking V, Selent U, Kohler E, Landgraf A, Wolfes H, Alves J . Mg2+ confers DNA binding specificity to the EcoRV restriction endonuclease. Biochemistry. 1992; 31(15):3727-32. DOI: 10.1021/bi00130a001. View

Zebala J, Choi J, BARANY F . Characterization of steady state, single-turnover, and binding kinetics of the TaqI restriction endonuclease. J Biol Chem. 1992; 267(12):8097-105. View

10.

Modrich P . Mechanisms and biological effects of mismatch repair. Annu Rev Genet. 1991; 25:229-53. DOI: 10.1146/annurev.ge.25.120191.001305. View

11.

Kong X, Onrust R, ODonnell M, Kuriyan J . Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell. 1992; 69(3):425-37. DOI: 10.1016/0092-8674(92)90445-i. View

12.

Selent U, Ruter T, Kohler E, Liedtke M, Thielking V, Alves J . A site-directed mutagenesis study to identify amino acid residues involved in the catalytic function of the restriction endonuclease EcoRV. Biochemistry. 1992; 31(20):4808-15. DOI: 10.1021/bi00135a010. View

13.

Vermote C, Halford S . EcoRV restriction endonuclease: communication between catalytic metal ions and DNA recognition. Biochemistry. 1992; 31(26):6082-9. DOI: 10.1021/bi00141a018. View

14.

Winkler F, Banner D, Oefner C, Tsernoglou D, Brown R, Heathman S . The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments. EMBO J. 1993; 12(5):1781-95. PMC: 413397. DOI: 10.2210/pdb4rve/pdb. View

15.

Heitman J . On the origins, structures and functions of restriction-modification enzymes. Genet Eng (N Y). 1993; 15:57-108. DOI: 10.1007/978-1-4899-1666-2_4. View

16.

Jeltsch A, Alves J, Wolfes H, Maass G, Pingoud A . Substrate-assisted catalysis in the cleavage of DNA by the EcoRI and EcoRV restriction enzymes. Proc Natl Acad Sci U S A. 1993; 90(18):8499-503. PMC: 47384. DOI: 10.1073/pnas.90.18.8499. View

17.

Vipond I, Halford S . Structure-function correlation for the EcoRV restriction enzyme: from non-specific binding to specific DNA cleavage. Mol Microbiol. 1993; 9(2):225-31. DOI: 10.1111/j.1365-2958.1993.tb01685.x. View

18.

Siksnys V, Pleckaityte M . Catalytic and binding properties of restriction endonuclease Cfr9I. Eur J Biochem. 1993; 217(1):411-9. DOI: 10.1111/j.1432-1033.1993.tb18260.x. View

19.

Jeltsch A, Fritz A, Alves J, Wolfes H, Pingoud A . A fast and accurate enzyme-linked immunosorbent assay for the determination of the DNA cleavage activity of restriction endonucleases. Anal Biochem. 1993; 213(2):234-40. DOI: 10.1006/abio.1993.1415. View

20.

Kabata H, Kurosawa O, Arai I, Washizu M, Margarson S, Glass R . Visualization of single molecules of RNA polymerase sliding along DNA. Science. 1993; 262(5139):1561-3. DOI: 10.1126/science.8248804. View