Analysis of the recognition mechanism involved in the EcoRV catalyzed cleavage of DNA using modified oligodeoxynucleotides

Fließ, Anja; Wolfes, Heiner; Seela, Frank; Pingoud, Alfred

doi:10.1093/nar/16.24.11781

Cited by 59 publications

(43 citation statements)

References 71 publications

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“…The EcoRV -DNA interface and in particular the recognition contacts have been probed with modified DNA substrates (Fliess et al, 1988;Mazzarelli et al, 1989;Newman et al, 1990) and with mutant proteins (Thielking et al, 1991;. There is very satisfactory agreement in the sense that modifications which directly perturb the observed interactions produce large reductions in the cleavage rates.…”

Section: Discussionmentioning

confidence: 92%

The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments.

et al. 1993

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The crystal structure of EcoRV endonuclease has been determined at 2.5 A resolution and that of its complexes with the cognate DNA decamer GGGATATCCC (recognition sequence underlined) and the non‐cognate DNA octamer CGAGCTCG at 3.0 A resolution. Two octamer duplexes of the non‐cognate DNA, stacked end‐to‐end, are bound to the dimeric enzyme in B‐DNA‐like conformations. The protein‐‐DNA interactions of this complex are prototypic for non‐specific DNA binding. In contrast, only one cognate decamer duplex is bound and deviates considerably from canonical B‐form DNA. Most notably, a kink of approximately 50 degrees is observed at the central TA step with a concomitant compression of the major groove. Base‐specific hydrogen bonds between the enzyme and the recognition base pairs occur exclusively in the major groove. These interactions appear highly co‐operative as they are all made through one short surface loop comprising residues 182–186. Numerous contacts with the sugar phosphate backbone extending beyond the recognition sequence are observed in both types of complex. However, the total surface area buried on complex formation is > 1800 A2 larger in the case of cognate DNA binding. Two acidic side chains, Asp74 and Asp90, are close to the reactive phosphodiester group in the cognate complex and most probably provide oxygen ligands for binding the essential cofactor Mg2+. An important role is also indicated for Lys92, which together with the two acidic functions appears to be conserved in the otherwise unrelated structure of EcoRI endonuclease. The structural results give new insight into the physical basis of the remarkable sequence specificity of this enzyme.

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Section: Discussionmentioning

confidence: 92%

The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments.

et al. 1993

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“…These analogues gave excellent substrates with kinetic properties very similar to the parent. Previously, it has been observed that dSMeC or the isosteric 5-bromodeoxycytidine gave good substrates for the endonuclease (Fliess et al, 1988). However, the role of the 4-amino group of dC has not been probed using analogues.…”

Section: ~( P G a C [~~~P ] A T A T C G T C )mentioning

confidence: 99%

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

Waters¹,

Connolly²

1994

Biochemistry

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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)

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“…Previous reports have shown that some restriction enzymes can recognize and cleave many chemically modified DNA substrates with certain hydrolysis efficiency . In most, if not all, of these cases, the modifications involving either base or sugar‐phosphate backbone tolerated by the restriction enzymes were induced on a single strand, rather than both strands, of the recognition site .…”

Section: Discussionmentioning

confidence: 99%

“…Binding to the specific recognition site with suitable flanking sequences will then activate the restriction enzyme by increasing catalysis . Synthetic modified oligodeoxyribonucleotide duplexes have been applied to probe the DNA determinants required for such a recognition by a series of restriction enzymes . The success of this approach depends mostly on the availability of a large number of suitably modified DNA substrates.…”

Section: Introductionmentioning

confidence: 99%

Sequence-dependent cleavage of mismatched DNA byBanI restriction endonuclease

2017

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Restriction enzymes have previously shown the ability to cleave DNA substrates with mismatched base(s) in recognition sequences; in this study, Ban I endonuclease demonstrated this same ability. Single base substitutions were introduced, and fragments containing various types of unpaired base(s) (heteroduplex fragments) within the Ban I endonuclease recognition sequence, 5'-G|GPyPuCC-3', were generated. Each of the heteroduplex fragments was treated with Ban I endonuclease and analyzed by denaturing gradient gel electrophoresis. Our results showed that heteroduplex fragments containing mismatched bases at either the first or third position of the Ban I recognition sequence or, because of the symmetrical structure of the sequence, the sixth or fourth position on the opposite strand were cleaved by the enzyme. Furthermore, these cleaved fragments contained at least one strand corresponding to the original Ban I recognition sequence. Fragments with mismatches formed by an A (noncanonical, nc) opposite a purine (canonical, ca) or a T (nc) opposite a pyrimidine (ca) were cleaved more efficiently than other types of mismatched bases. These results may help elucidate the mechanisms by which DNA and protein interact during the process of DNA cleavage by Ban I endonuclease.

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Analysis of the recognition mechanism involved in the EcoRV catalyzed cleavage of DNA using modified oligodeoxynucleotides

Cited by 59 publications

References 71 publications

The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments.

The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments.

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

Sequence-dependent cleavage of mismatched DNA byBanI restriction endonuclease

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