Interaction of the Restriction Endonuclease EcoRV with the Deoxyguanosine and Deoxycytidine Bases in Its Recognition Sequence

Abstract

The interaction of the EcoRV restriction endonuclease with the dG and dC bases in its recognition sequence (GATATC) has been studied using base analogues. These modified dG and dC bases each have a single potential protein contact removed. The analogues have been incorporated into the self-complementary dodecamer d(pGACGATATCGTC) at the appropriate positions (underlined). Many of the analogues caused no change in the Tm of the duplex or else lowered the Tm by a small amount such that a duplex was still formed at temperatures suitable for enzyme assay. However, the dG analogue 2-aminopurine-1-beta-D-2'-deoxyriboside destabilized the duplex to such an extent that the 12'-mer could not be used for enzyme assays. To overcome this, a longer self-complementary 18'-mer was used with this modified base. The circular dichroism spectra of the modified base containing 12'-mers (and the 18'-mer in the case of 2-aminopurine) were very similar to the parent sequences lacking modified bases. This demonstrates the formation of B-DNA structures in all cases and similar overall conformations. The Km and kcat values for the various modified oligomers have been determined, and these data have been used to assess the roles that functional groups on the dG and dC bases play in the recognition and hydrolysis of GATATC sequences by the endonuclease. The results obtained here have been compared to the crystal structures of the EcoRV complexed with a GATATC sequence, and this has allowed a critical evaluation of the base analogue approach.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific binding by EcoRV endonuclease to its DNA recognition site GATATC

“…In the cognate complex, the protein makes extensive hydrogen-bonding contacts with the bases in the DNA major groove; these contacts are absent in the non-cognate complex and the corresponding protein elements are largely disordered. The observed protein-base contacts are generally consistent with those inferred from biochemical studies on the effects of modi®ed DNA bases (Mazzarelli et al, 1989;Newman et al 1990b;Waters & Connolly, 1994). In summary, the cognate complex in the absence of Mg 2 appears to have all the structural properties expected for a speci®c recognition complex, but the non-cognate complex does not.…”

The EcoRV Modification Methylase Causes Considerable Bending of DNA upon Binding to Its Recognition Sequence GATATC

“…Restriction enzymes were purchased from NBL (Northumbria Biological Laboratories, Cramlington, UK) and Taq DNA polymerase was from Boehringer Mannheim (Lewes, UK). Oligonucleotides primers for PCR amplification were synthesized on an Applied Biosystems 381A DNA synthesizer using the phosphoramidite method (Newman et al, 1990;Connolly 1991Connolly , 1992Waters and Connolly, 1994) with reagents from either Cruachem (Glasgow, Scotland) or Applied Biosystems (Warrington, UK). The phosphoramidite of 6-methyldeoxyadenosine was purchased from Pharmacia.…”

Use of plasmon coupling to reveal the dynamics of DNA bending and cleavage by single EcoRV restriction enzymes

“…The similarity of the two rate constants is in agreement with the assertion that the trajectories comprise molecular events like DNA bending, DNA hydrolysis, and product release, but not DNA binding, which is expected to be much slower in case of the 30-bp spacer because of steric hindrance. The catalytic cycle of EcoRV with short (7-14 bp) substrate DNAs has been investigated in bulk by using FRET, stopped-flow fluorescence, fluorescence anisotropy, and quench-flow methods (46,48,49). These studies found that the turnover rate k cat is limited by rates of DNA hydrolysis and product release (Fig.…”