Interaction of the EcoRV restriction endonuclease with the deoxyadenosine and thymidine bases in its recognition hexamer d(GATATC)

Newman, Patrick C.; Williams, David M.; Cosstick, Richard; Seela, Frank; Connolly, Bernard A.

doi:10.1021/bi00494a021

Cited by 77 publications

(52 citation statements)

References 38 publications

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“…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.…”

Section: Introductionsupporting

confidence: 77%

Specific binding by EcoRV endonuclease to its DNA recognition site GATATC

Engler

Welch

Jen‐Jacobson

1997

Journal of Molecular Biology

143

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Section: Introductionsupporting

confidence: 77%

Specific binding by EcoRV endonuclease to its DNA recognition site GATATC

Engler

Welch

Jen‐Jacobson

1997

Journal of Molecular Biology

143

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“…We have observed a similar improvement in binding at position 12 For this protein-DNA complex, we have presented evidence that supports hydrogen bonding contact sites at positions that are predicted to be directly involved in the formation of the protein-DNA interface. We have also demonstrated that the central region of the operator is responsible, in part, for modulating the protein-DNA complex indirectly and by manipulating these indirect effects with 'anomalous' base pairs a protein-DNA complex more stable than the wild-type operatorrepressor was achieved.…”

Section: The Role Of the Central Region Of The Operatorsupporting

confidence: 69%

Modified nucleotides reveal the indirect role of the central base pairs in stabilizing thelacrepressor-operator complex

Zhang

Gottlieb

1995

Nucl Acids Res

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Guanine residues in the lac operator were replaced by 2-aminopurine or purine analogues, pairing the modified nucleotides with C. The observed equilibrium dissociation constants for lac repressor binding to substituted operators were measured in 10 mM Tris, 150 mM KCI, 0.1 mM EDTA, 0.1 mM DTE, pH 7.6 at 250C. These measurements revealed five positions that destabilized the complex when substituted with either analogue. Two positions, which are related by a 2-fold symmetry, are in the major groove of the operator thought to directly interact with the protein. Three sites were in the central region of the operator. A purine analogue at a sixth site perturbed the local DNA structure and destabilized the complex. Alkylation interference experiments of the 2-aminopurine substituted operators demonstrated that, of the five affected, two substitutions displayed altered phosphate interference patterns at the phosphate adjacent to the substituted base. For these operators, complex formation was measured in different concentrations of KCI to assess the contribution of counterion release to the bimolecular process. The results indicated that both complexes were similar to wild-type, although minor changes were observed. The Iobs of the complex was then measured when 2-aminopurine or purine analogues were paired with uracil nucleotide, a base pair that serves to stabilize the DNA. The introduction of the new base pairs revealed two effects on the bimolecular interaction. For those operator sites that are thought to perturb the interaction directly, the affinity of the complex was weakened to levels observed for the singly-substituted operators. In contrast, the nucleotides of 2-aminopurine paired with uracil positioned in the central region of the operator served to enhance the stability of the complex. The purine-uracil base pair substitution op the other hand had a significant destabilizing effect on the interaction. We propose that the central base pairs modulate binding of the complex by altering the intrinsic properties of the DNA. Two specific attributes are required to achieve the lowest free energy of interaction. The DNA must have two interstrand hydrogen bonds to stabilize the duplex and it must have properties associated with directional bending or unwinding. This analysis does not rule out contributions by direct interactions between the protein and the central region of the operator but underscores how indirect effects play a major role in complex formation in this system.

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“…4 nmol (20 pM) of double-stranded oligodeoxynucleotide (not phosphorylated at the 5'-position) was incubated in 200 p L of 50 mM Hepes, pH 7.5, 10 mM MgC12, 100 mM NaCl, and 10 units (manufacturers units, special grade for molecular biology, obtained from Promega, Southampton, U.K.) of alkaline phosphatase in the presence of 340 pmol (1.7 pM) of endonuclease dimer. The reaction was monitored by reversephase HPLC (Newman et al, 1990a). The alkaline phosphatase removes the 5'-phosphate from one of the products, and this eases separation of the two product oligodeoxynucleotides.…”

Section: Methodsmentioning

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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Interaction of the EcoRV restriction endonuclease with the deoxyadenosine and thymidine bases in its recognition hexamer d(GATATC)

Cited by 77 publications

References 38 publications

Specific binding by EcoRV endonuclease to its DNA recognition site GATATC

Specific binding by EcoRV endonuclease to its DNA recognition site GATATC

Modified nucleotides reveal the indirect role of the central base pairs in stabilizing thelacrepressor-operator complex

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

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