Interaction of the EcoRV restriction endonuclease with the deoxyadenosine and thymidine bases in its recognition hexamer d(GATATC).
A set of dA and T analogues suitable for the study of protein DNA interactions have been incorporated into the central d(ATAT) sequence within d(GACGATATCGTC). The individual analogues have one potential protein contact (either a hydrogen-bonding group or a CH3 group capable of a van der Waals interaction) deleted. In general, the modified bases do not perturb the overall structure of the dodecamer, enabling results obtained to be simply interpreted in terms of loss of protein DNA contacts. We have used the modified oligodeoxynucleotide set to study the recognition of DNA by the EcoRV restriction endonuclease [recognition sequence d(GATATC)]. The kcat and Km values for the set have been determined, and a comparison with results seen with the parent oligodeoxynucleotide (containing no modified bases) has been carried out. Three classes of results are seen. First, some analogues lead to no change in kinetic parameters, meaning no enzyme contact at the altered site. Second (this is seen for most of the modified oligodeoxynucleotides), a drop in the kcat/Km ratio relative to the parent is observed. This comes mainly from a decrease in kcat, implying that the endonuclease uses the interaction under study to lower the transition-state barrier rather than to bind the substrate. Analyses of these results show that the drop in kcat/Km is what would be expected for the simple loss of a hydrogen bond or a CH3 contact between the enzyme and the oligodeoxynucleotide. This implies a contact of these types at these sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Newman
P C
PC
Department of Biochemistry (SERC Molecular Recognition Centre), University of Southampton, U.K.
Williams
D M
DM
Cosstick
R
R
Seela
F
F
Connolly
B A
BA
D000241:Adenosine / Q000031:analogs & derivatives; D001483:Base Sequence; D003839:Deoxyadenosines / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity; D013936:Thymidine / Q000031:analogs & derivatives / Q000378:metabolism*
D003839:Deoxyadenosines; D009838:Oligodeoxyribonucleotides; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D000241:Adenosine; D013936:Thymidine