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Biomedical subjects

I B Vipond

Publications and source records attributed to I B Vipond.

25 records · Page 2Linked to original sources
PubMed1995

Rapid reaction analysis of the catalytic cycle of the EcoRV restriction endonuclease.

We have used the intrinsic tryptophan fluorescence of the EcoRV restriction endonuclease to monitor changes in protein conformation during binding and cleavage of a duplex oligodeoxynucleotide substrate. Appropriate conditions for single-turnover reactions were first determined by steady-state kinetics. When single turnovers were monitored by stopped-flow fluorescence, the mixing together of EcoRV, oligonucleotide and MgCl2 resulted in a rapid increase in tryptophan fluorescence followed by a slow decrease. Further analysis by order-of-mixing and quench experiments showed that the transient increase in fluorescence was due to a conformational change coupled to DNA binding, while the subsequent decay was concomitant with phosphodiester hydrolysis. The rate of the latter step varied with the concentration of Mg2+ ions, but another Mg(2+)-dependent transition was observed upon the addition of MgCl2 to a preformed enzyme-DNA complex. These results lead to a reaction scheme in which one Mg2+ binds to the active site prior to phosphodiester hydrolysis but a second Mg2+ is then needed to carry out the hydrolytic reaction. This scheme is correlated to the crystal structures of the EcoRV endonuclease and its complexes with DNA and Mg2+ ions.

Base Sequence↗
PubMed1993

RNA editing in Newcastle disease virus.

The co-transcriptional editing of the Newcastle disease virus (NDV) P gene has been studied by sequence analysis of cloned viral genomic RNA and mRNA. Evidence has been obtained for the specific insertion of non-templated G nucleotides, the consequence of which is the generation of three populations of P gene-derived mRNAs. The three populations encode proteins (P, V and W) which have a common N-terminal region, but which utilize three different reading frames at their C termini. Paradoxically, NDV edits its P gene mRNA by the insertion of non-templated G residues in a manner similar to Sendai and measles viruses (P-->V editing) despite its apparent closer evolutionary relationship to the simian virus type 5, mumps and related group of viruses which edit a V genomic sequence to generate an mRNA to encode a functional P protein (V-->P editing).

Amino Acid Sequence↗
PubMed1993

Structure-function correlation for the EcoRV restriction enzyme: from non-specific binding to specific DNA cleavage.

The EcoRV restriction endonuclease cleaves DNA at its recognition sequence at least a million times faster than at any other DNA sequence. The only cofactor it requires for activity is Mg2+; but in binding to DNA in the absence of Mg2+, the EcoRV enzyme shows no specificity for its recognition site. Instead, the reason why EcoRV cuts one DNA sequence faster than any other is that the rate of cleavage is controlled by the binding of Mg2+ to EcoRV-DNA complexes: the complex at the recognition site has a high affinity for Mg2+, while the complexes at other DNA sequences have low affinities for Mg2+. The structures of the EcoRV endonuclease, and of its complexes with either specific or non-specific DNA, have been solved by X-ray crystallography. In the specific complex, the protein interacts with the bases in the recognition sequence and the DNA takes up a highly distorted structure. In the non-specific complex with an unrelated DNA sequence, there are virtually no interactions with the bases and the DNA retains a B-like structure. Since the free energy changes for the formation of specific and non-specific complexes are the same, the energy from the specific interactions balances that required for the distortion of the DNA. The distortion inserts the phosphate at the scissile bond into the active site of the enzyme, where it forms part of the binding site for Mg2+. Without this distortion, the EcoRV-DNA complex would be unable to bind Mg2+ and thus unable to cleave DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

EcoRV restriction endonuclease: communication between DNA recognition and catalysis.

A genetic system was constructed for the mutagenesis of the EcoRV restriction endonuclease and for the overproduction of mutant proteins. The system was used to make two mutants of EcoRV, with Ala in place of either Asn185 or Asn188. In the crystal structure of the EcoRV-DNA complex, both Asn185 and Asn188 contact the DNA within the EcoRV recognition sequence. But neither mutation affected the ability of the protein to bind to DNA. In the absence of metal ion cofactors, the mutants bound DNA with almost the same affinity as that of the wild-type enzyme. In the presence of Mg2+, both mutants retained the ability to cleave DNA specifically at the EcoRV recognition sequence, but their activities were severely depressed relative to that of the wild-type. In contrast, with Mn2+ as the cofactor, the mutant enzymes cleaved the EcoRV recognition site with activities that were close to that of the wild-type. When bound to DNA at the EcoRV recognition site, the mutant proteins bound Mn2+ ions readily, but they had much lower affinities for Mg2+ ions than the wild-type enzyme. This was the reason for their low activities with Mg2+ as the cofactor. The arrangement of the DNA recognition functions, at one location in the EcoRV restriction enzyme, are therefore responsible for organizing the catalytic functions at a separate location in the protein.

Asparagine↗