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|Title:||Use of Oligo - Deoxyribonucleotides Containing Base Analogues to Study the Sequence Specificity of the Eco R1 Restriction Endonuclease and Modification Methylase|
|Author(s):||Brennan, Catherine Anne|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Models have proposed that recognition of specific DNA sequences by proteins is accomplished by specific hydrogen bond formation between the protein and groups accessible in the major and minor grooves of the DNA. We have examined DNA-protein interactions involved in the recognition of a specific DNA sequence by the EcoR1 restriction and modification enzymes by using base analogues to change functional groups in the major and minor grooves of the DNA. The EcoR1 system consists of an endonuclease that cleaves the DNA sequence, GAATTC, between the guanine and adenine residues and a methylase that methylates the 6-amino group of the central adenine residue. Methylation of the recognition sequence prevents cleavage of the site by the endonuclease. The EcoR1 system is a convenient system for the study of sequence specific DNA-protein interactions because both enzymes have been purified and well characterized and show a high specificity for their recognition sequence. In addition, the recognition sequence is short and self-complementary oligodeoxyribonucleotides that are only eight base pairs long and contain the recognition sequence are substrates for the enzymes thereby allowing simple substrates and their analogues to be examined.
Self-complementary oligodeoxyribonucleotides with base analogues in the EcoR1 recognition sequence were synthesized by a general method that allows incorporation of the analogues at specific positions in the sequence. This method uses chemistry to synthesize partial sequences but circumvents the need for blocked base analogues by incorporating the analogues enzymatically. T4 RNA ligase was used to add base analogues to the oligodeoxynucleotides and yields from 54 to greater than 95% were obtained. The products of these reactions were reactive in subsequent RNA ligase joining reactions thus allowing sequential syntheses of oligodeoxynucleotides with base analogues incorporated at specific positions. Oligodeoxynucleotides were joined to oligodeoxynucleotides containing analogues at their 3' terminus in yields from 22 to 78%.
The effects of the base analogue substitutions on the interactions between the EcoR1 enzymes and their recognition sequence were examined by determining the steady state kinetic parameters of the enzymes with the octanucleotide analogues as substrates. By a comparison of the reactivity of the octanucleotide analogues to a control octanucleotide, we have identified groups on the DNA that may be involved in site recognition and catalysis by each of the EcoR1 enzymes.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1985.
|Date Available in IDEALS:||2014-12-15|