Biomedical subjects

Merlind Mücke

Publications and source records attributed to Merlind Mücke.

4 recordsLinked to original sources

PubMed2004

Crystal structure of type IIE restriction endonuclease EcoRII reveals an autoinhibition mechanism by a novel effector-binding fold.

EcoRII is a type IIE restriction endonuclease that interacts with two copies of the DNA recognition sequence 5'CCWGG, one being the actual target of cleavage, the other serving as the allosteric effector. The mode of enzyme activation by effector binding is unknown. To investigate the molecular basis of activation and cleavage mechanisms by EcoRII, the crystal structure of EcoRII mutant R88A has been solved at 2.1A resolution. The EcoRII monomer has two domains linked through a hinge loop. The N-terminal effector-binding domain has a novel DNA recognition fold with a prominent cleft. The C-terminal catalytic domain has a restriction endonuclease-like fold. Structure-based sequence alignment identified the putative catalytic site of EcoRII that is spatially blocked by the N-terminal domain. The structure together with the earlier characterized EcoRII enzyme activity enhancement in the absence of its N-terminal domain reveal an autoinhibition/activation mechanism of enzyme activity mediated by a novel effector-binding fold. This is the first case of autoinhibition, a mechanism described for many transcription factors and signal transducing proteins, of a restriction endonuclease.

Allosteric Regulation↗

PubMed2002

EcoRII: a restriction enzyme evolving recombination functions?

The restriction endonuclease EcoRII requires the cooperative interaction with two copies of the sequence 5'CCWGG for DNA cleavage. We found by limited proteolysis that EcoRII has a two-domain structure that enables this particular mode of protein-DNA interaction. The C-terminal domain is a new restriction endonuclease, EcoRII-C. In contrast to the wild-type enzyme, EcoRII-C cleaves DNA specifically at single 5'CCWGG sites. Moreover, substrates containing two or more cooperative 5'CCWGG sites are cleaved much more efficiently by EcoRII-C than by EcoRII. The N-terminal domain binds DNA specifically and attenuates the activity of EcoRII by making the enzyme dependent on a second 5'CCWGG site. Therefore, we suggest that a precursor EcoRII endonuclease acquired an additional DNA-binding domain to enable the interaction with two 5'CCWGG sites. The current EcoRII molecule could be an evolutionary intermediate between a site-specific endonuclease and a protein that functions specifically with two DNA sites such as recombinases and transposases. The combination of these functions may enable EcoRII to accomplish its own propagation similarly to transposons.

Base Sequence↗

PubMed2002

Counting CAG repeats in the Huntington's disease gene by restriction endonuclease EcoP15I cleavage.

Huntington's disease (HD) is a progressive neurodegenerative disorder with autosomal-dominant inheritance. The disease is caused by a CAG trinucleotide repeat expansion located in the first exon of the HD gene. The CAG repeat is highly polymorphic and varies from 6 to 37 repeats on chromosomes of unaffected individuals and from more than 30 to 180 repeats on chromosomes of HD patients. In this study, we show that the number of CAG repeats in the HD gene can be determined by restriction of the DNA with the endonuclease EcoP15I and subsequent analysis of the restriction fragment pattern by electrophoresis through non-denaturing polyacrylamide gels using the ALFexpress DNA Analysis System. CAG repeat numbers in the normal (30 and 35 repeats) as well as in the pathological range (81 repeats) could be accurately counted using this assay. Our results suggest that this high-resolution method can be used for the exact length determination of CAG repeats in HD genes as well as in genes affected in related CAG repeat disorders.

DNA Mutational Analysis↗

PubMed2002

Asymmetric photocross-linking pattern of restriction endonuclease EcoRII to the DNA recognition sequence.

The EcoRII homodimer engages two of its recognition sequences (5'-CCWGG) simultaneously and is therefore a type IIE restriction endonuclease. To identify the amino acids of EcoRII that interact specifically with the recognition sequence, we photocross-linked EcoRII with oligonucleotide substrates that contained only one recognition sequence for EcoRII. In this recognition sequence, we substituted either 5-iododeoxycytidine for each C or 5-iododeoxyuridine for A, G, or T. These iodo-pyrimidine bases were excited using a UV laser to result in covalent cross-linking products. The yield of EcoRII photocross-linked to the 5'-C of the 5'-CCAGG strand of the recognition sequence was 45%. However, we could not photocross-link EcoRII to the 5'-C of the 5'-CCTGG strand. Thus, the contact of EcoRII to the bases of the recognition sequence appears to be asymmetric, unlike that expected for most type II restriction endonucleases. Tryptic digestion of free and of cross-linked EcoRII, followed by high performance liquid chromatography (HPLC) separation of the individual peptides and Edman degradation, identified amino acids 25-49 of EcoRII as the cross-linking peptide. Mutational analysis of the electron-rich amino acids His(36) and Tyr(41) of this peptide indicates that Tyr(41) is the amino acid involved in the cross-link and that it therefore contributes to specific DNA recognition by EcoRII.

Base Sequence↗