Abstract

The detailed structural and energetic origins of sequence-specificity in the recognition and modification of DNA by proteins will be investigated using EcoRV endonuclease as the experimental system. A strategy encompassing directed modification of the enzyme and DNA, based on the high-resolution Xray cocrystal structures, will be used to test current hypotheses regarding the basis for discrimination. Specific questions to be addressed include the role of extensive enzyme and DNA conformational changes (induced fit), which are required to form the specific complex. The underlying basis for indirect readout of DNA sequence information will also be addressed. Rational strategies will be employed to expand the sequence specificity of the enzyme beyond the six base-pair cognate DNA site, and the database of known restriction endonuclease-DNA structures will be expanded by determination of several new enzymes. Creation of novel restriction endonucleases with expanded specificity will be extremely useful in aiding current efforts at large-scale physical mapping of the human genome. Since restriction endonucleases form an essential part of the recombinant DNA technology underlying all areas of biological research, the development of enzymes with new specificities will have a wide-ranging impact on the development of therapies for many human diseases.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM053763-07
Application #
6519702
Study Section
Biochemistry Study Section (BIO)
Program Officer
Jones, Warren
Project Start
1996-03-01
Project End
2005-02-28
Budget Start
2002-03-01
Budget End
2005-02-28
Support Year
7
Fiscal Year
2002
Total Cost
$221,695
Indirect Cost

  Institution

Name
University of California Santa Barbara
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Santa Barbara
State
CA
Country
United States
Zip Code
93106

  Publications

Menezes, Sheena; Gaston, Kirk W; Krivos, Kady L et al. (2011) Formation of m2G6 in Methanocaldococcus jannaschii tRNA catalyzed by the novel methyltransferase Trm14. Nucleic Acids Res 39:7641-55
Hancock, Stephen P; Hiller, David A; Perona, John J et al. (2011) The energetic contribution of induced electrostatic asymmetry to DNA bending by a site-specific protein. J Mol Biol 406:285-312
Hou, Ya-Ming; Perona, John J (2010) Stereochemical mechanisms of tRNA methyltransferases. FEBS Lett 584:278-86
Christian, Thomas; Lahoud, Georges; Liu, Cuiping et al. (2010) Mechanism of N-methylation by the tRNA m1G37 methyltransferase Trm5. RNA 16:2484-92
Estabrook, R August; Nguyen, Trung T; Fera, Nickolas et al. (2009) Coupling sequence-specific recognition to DNA modification. J Biol Chem 284:22690-6
Coffin, Stephanie R; Reich, Norbert O (2008) Modulation of Escherichia coli DNA methyltransferase activity by biologically derived GATC-flanking sequences. J Biol Chem 283:20106-16
Shieh, Fa-Kuen; Reich, Norbert O (2007) AdoMet-dependent methyl-transfer: Glu119 is essential for DNA C5-cytosine methyltransferase M.HhaI. J Mol Biol 373:1157-68
Youngblood, Ben; Shieh, Fa-Kuen; Buller, Fabian et al. (2007) S-adenosyl-L-methionine-dependent methyl transfer: observable precatalytic intermediates during DNA cytosine methylation. Biochemistry 46:8766-75
Hiller, David A; Perona, John J (2006) Positively charged C-terminal subdomains of EcoRV endonuclease: contributions to DNA binding, bending, and cleavage. Biochemistry 45:11453-63
Youngblood, Ben; Buller, Fabian; Reich, Norbert O (2006) Determinants of sequence-specific DNA methylation: target recognition and catalysis are coupled in M.HhaI. Biochemistry 45:15563-72

Showing the most recent 10 out of 22 publications