Communication between protein and DNA is one of the most fundamental processes in all living cells. To understand the structural basis for this process in the Eschericia coli RI restriction-modification system, one needs to know the three-dimensional structures of all the players involved: they are the structures of free DNAs with and without the recognition sequence, the free endonuclease, the free methylase, the complexes between the endonuclease and DNA with and without the cognate sequence, and the corresponding complexes with the methylase. Of these eight structures only two, a free DNA containing the recognition sequence and a recognition complex, have been determined so far by X-ray crystallographic methods. Our overall objective is to determine the three-dimensional structures of the remaining six structures plus the recognition complexes with different flanking sequences. We would like to establish the structural basis for understanding the DNA sequence specific/non-specific interaction process and the dependence of the recognition on the flanking sequences at atomic resolution. We have constructed an over-producing strain of E. coli for the production of the endonuclease and methylase, from which we have prepared gram quantities of the enzymes. Several DNA sequences with and without the cognate sequence have also been synthesized. From these have been obtained single crystals of one DNA sequence, the endonuclease alone, and the complex between the endonuclease and a cognate DNA. X-ray crystallographic studies on these crystals and crystallization of the others are in progress.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM029287-06
Application #
3276843
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1981-08-01
Project End
1990-04-30
Budget Start
1987-05-01
Budget End
1988-04-30
Support Year
6
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Type
Schools of Arts and Sciences
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Glaeser, R M; Tong, L; Kim, S H (1989) Three-dimensional reconstructions from incomplete data: interpretability of density maps at ""atomic"" resolution. Ultramicroscopy 27:307-18
Pearlman, D A; Kim, S H (1988) Conformational studies of nucleic acids. V. Sequence specificities in the conformational energetics of oligonucleotides: the homo-tetramers. Biopolymers 27:59-77
de Vos, A M; Tong, L; Milburn, M V et al. (1988) Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21. Science 239:888-93
Jancarik, J; de Vos, A; Kim, S H et al. (1988) Crystallization of human c-H-ras oncogene products. J Mol Biol 200:205-7
Kim, S H; de Vos, A M; Tong, L et al. (1988) ras oncogene proteins: three-dimensional structures, functional implications, and a model for signal transducer. Cold Spring Harb Symp Quant Biol 53 Pt 1:273-81
Kim, S H; Cech, T R (1987) Three-dimensional model of the active site of the self-splicing rRNA precursor of Tetrahymena. Proc Natl Acad Sci U S A 84:8788-92
Pearlman, D A; Kim, S H (1986) Conformational studies of nucleic acids: III. Empirical multiple correlation functions for nucleic acid torsion angles. J Biomol Struct Dyn 4:49-67
Pearlman, D A; Kim, S H (1986) Conformational studies of nucleic acids: IV. The conformational energetics of oligonucleotides: d(ApApApA) and ApApApA. J Biomol Struct Dyn 4:69-98
Holbrook, S R; Wang, A H; Rich, A et al. (1986) Local mobility of nucleic acids as determined from crystallographic data. II. Z-form DNA. J Mol Biol 187:429-40
Kim, S H; Pearlman, D A; Holbrook, S R et al. (1985) Structures of DNA containing psoralen crosslink and thymine dimer. Prog Clin Biol Res 172A:143-52

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