Investigation of the sequence specific DNA/protein interactions of the ECORV restriction enzyme

Newman, Patrick Charles (1989) Investigation of the sequence specific DNA/protein interactions of the ECORV restriction enzyme. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

The EcoRV restriction enzyme from Escherichia coli recognises the sequence GATATC on double stranded DNA and cuts between the central residues with very high specificity. The self complementary dodecadeoxynucleotide dGACGATATCGTC which contains the EcoRV recognition site (underlined) is also a substrate for the endonuclease. The thesis describes the synthesis of fourteen dodecamers of the above parent sequence in which the functional groups of the central ATAT residues accessible to the protein via the DNA major and minor grooves have been systematically and sequentially deleted. Conservative contact deletions were achieved by the substitution of the two deoxyadenosine residues in turn with the base analogues purine deoxyriboside (dP), 7-deazadeoxyadenosine (d^7CA) and 3-deazadeoxyadenosine (d^3CA). Similarly the two thymidine residues were substituted with deoxyuridine (dU), 5-methyl-2-pyrimidonone deoxyriboside (d^4HT),4-thiothymidine (d^4ST) and 2-thiothymidine (d^2ST). To obtain the complete set of analogues, efficient synthetic routes for the formation of derivatives of d^4HT, d^4ST and d^2ST suitable for oligodeoxynucleotide synthesis using the cyanoethyl phosphoramidite approach are described (also see Connolly & Newman (1989) Nucleic Acids Research 17, 4957-4974). The work represents the first time that d^4ST and d^2ST have been incorporated into synthetic oligodeoxynucleotides. All the analogue substituted dodecamers were obtained in a pure state and characterisation by deoxynucleoside composition analysis, hyperchromicity, melting temperature and circular dichroism spectroscopy showed them to be of the expected composition and that they form stable duplexes of the B-DNA conformation. The only discrepancy is the circular dichroism spectra of the d^2ST dodecamers, which are not typical of B-DNA. Ten of the fourteen analogue substituted dodecamers were determined to be substrates for the EcoRV endonuclease. The kinetics of cleavage of these substrate dodecamers were analysed along with the parent dodecamer by assays utilising HPLC and gel separation methods, the latter method using 5'- phosphorylated substrates. It is found that 1: 5'-phosphorylation further decreases the cleavage rate of the most poorly cleaved analogue dodecamers, 2: the Mg²⁺ cofactor affinities for the parent dodecamer and two poorly cleaved substrates are similar, 3: the specificity of cleavage of the dodecamers (as determined by the k_cal/K_mM ratio) is 1000-fold lower than for the cleavage of plasmid DNA. Only two of the functional groups within the ATAT core of the GATATC site are not involved in recognition interactions (the 2-keto oxygen of the first thymidine and the N7 atom of the second deoxyadenosine). Apparent binding energy values for the deletion of the other functional groups are in the range of -4.8 to -6.7kJmol^-1 for the hydrogen bond donors/acceptors and -7.8 and -9.4kJmol^-1 for the two thymidine methyl groups. These values correspond to expected strengths of hydrogen bonds and Van der Waals contacts resepctively. It is concluded that EcoRV endonuclease makes sequence specific interactions to the majority of the functional groups of the ATAT core of GATATC both via the major and minor groove, many more than required for unambiguous discrimination of cognate and non-cognate sites.