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 synthesize defined sequences of DNA. We will continue to examine the effect upon the joining of oligodeoxyribonucleotides, under various reaction conditions, when the oligomer bearing the 3'- hydroxyl has a terminal ribonucleotide. The second major aim of these studies is to contribute to our understanding of specific protein-nucleic acid interactions by synthesizing a series of oligodeoxyribonucleotides containing variations of the EcoRI restriction endonuclease and methylase recognition sequence, d(GAATTC). Individual sequences will contain single or double base-analogue substitutions at defined sites. Analogues containing Hyp, 2-AminoPurine, 2,6-Diaminopurine, N6-MeAde, 3- deazaAde, Ura, 5-BrUra, 5-MeCyt, 5-BrCyt, 7-deazaGua, and 7- deazaAde will allow systematic alteration of many of the possible contact points between the enzymes and the major and minor grooves of the DNA. The EcoRI endonuclease and methylase will be examined by steady state kinetics and equilibrium binding techniques for their ability to interact with the modified sequences. In addition, we will purify, clone, sequence, and overeexpress and isoschizomer of EcoRI endonuclease and its cognate methylase from Rhodopseudomonas sphaeroides. These two enzymes, along with those from E. coli, will give us four sequence-specific proteins all recognizing the same sequence and will allow us to compare their properties.
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