Abstract

This research has two major aims. The first is to enhance the utility of T4 RNA ligase as a reagent for the synthesis and modification of nucleic acids by enlarging our understanding of its enzymological properties. a particular goal is to improve its ability to use DNA substrates so that it can be used to synthsize defined sequences of DNA. We propose to analyze reaction mixtures for each of the nucleotide, oligonucleotide, and enzyme species present under various conditions. The second major aim is to contribute to our understanding of specific protein-nucleic acid interactions by synthesizing a series of oligodeosyribonucleotides containing variations of the Eco RI restriction endonuclease and methylase recognition sequence, d(G-A-A-T-T-C). Individual syntherized sequences will contain single base analogue substitutions at define sites. Incorporation of Ura, 5-BrUra, 5-Mecyt, 5-BrCyt, 2-aminopurine, 2,6-diaminopurine, N(6)-MeAde, 3-deazaAde, and 7-deaza-8-azaGua will allow systematic alteration of may of the possible contact points between the enzymes and the major and minor grooves of the DNA. The two enzymes will be examined for their ability to interact with the modified substrates.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025621-07
Application #
3273158
Study Section
(MG)
Project Start
1978-12-01
Project End
1986-11-30
Budget Start
1984-12-01
Budget End
1985-11-30
Support Year
7
Fiscal Year
1985
Total Cost
Indirect Cost

  Institution

Name
University of Illinois Urbana-Champaign
Department
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820

  Publications

Thomas, Chad B; Gumport, Richard I (2006) Dimerization of the bacterial RsrI N6-adenine DNA methyltransferase. Nucleic Acids Res 34:806-15
Thomas, Chad B; Scavetta, Robert D; Gumport, Richard I et al. (2003) Structures of liganded and unliganded RsrI N6-adenine DNA methyltransferase: a distinct orientation for active cofactor binding. J Biol Chem 278:26094-101
Szegedi, S S; Gumport, R I (2000) DNA binding properties in vivo and target recognition domain sequence alignment analyses of wild-type and mutant RsrI [N6-adenine] DNA methyltransferases. Nucleic Acids Res 28:3972-81
Szegedi, S S; Reich, N O; Gumport, R I (2000) Substrate binding in vitro and kinetics of RsrI [N6-adenine] DNA methyltransferase. Nucleic Acids Res 28:3962-71
Scavetta, R D; Thomas, C B; Walsh, M A et al. (2000) Structure of RsrI methyltransferase, a member of the N6-adenine beta class of DNA methyltransferases. Nucleic Acids Res 28:3950-61
Fisher, E W; Yang, M T; Jeng, S T et al. (1995) Selection of mutations altering specificity in restriction-modification enzymes using the bacteriophage P22 challenge-phage system. Gene 157:119-21
Purmal, A A; Shabarova, Z A; Gumport, R I (1992) A new affinity reagent for the site-specific, covalent attachment of DNA to active-site nucleophiles: application to the EcoRI and RsrI restriction and modification enzymes. Nucleic Acids Res 20:3713-9
Van Cleve, M D; Gumport, R I (1992) Influence of enzyme-substrate contacts located outside the EcoRI recognition site on cleavage of duplex oligodeoxyribonucleotide substrates by EcoRI endonuclease. Biochemistry 31:334-9
Kaszubska, W; Webb, H K; Gumport, R I (1992) Purification and characterization of the M.RsrI DNA methyltransferase from Escherichia coli. Gene 118:5-11
Aiken, C R; Fisher, E W; Gumport, R I (1991) The specific binding, bending, and unwinding of DNA by RsrI endonuclease, an isoschizomer of EcoRI endonuclease. J Biol Chem 266:19063-9

Showing the most recent 10 out of 18 publications