Objective
The recently discovered electron microscopy (EM) approach for the observation of sequence-specific interaction of double stranded DNA (dsDNA) with different ligands such as methyltransferases, 5'-biotinylated purine and pyrimidine oligonucleotides forming intermolecular triplexes (TFO), peptide nucleic acids (PNA) and RecA-covered short oligonucleotides has been suggested as an effective method for the EM study of sequence-specific interaction of different ligands with dsDNA. It was also proposed that this approach could be intensively used for the localisation of the sites of known sequences alongside dsDNA molecules thus providing the tool for the physical mapping of DNA molecules and genomes, which is based on a precise knowledge of the particular site-selective interaction.
The significant advantage of the EM approaches stems from the ability to determine the molecular parameter inherent to the site-specific interaction and follow the local changes in DNA structures within or nearby to the specific complex. These changes in many cases are detected as DNA bending or kinks, which is now well understood as another very important parameter of the site-specific interaction. The target sequences which can be EM mapped might have regulatory meaning. For example, homopurine-homopyrimidine sequences are abundant in eucariotic genomes and little is known about their real distribution; long AT stretches are often present in the intergens regions and presumably can serve as structural elements which regulate genes expression. On the other hand, methyltransferases and especially synthetic TFO, PNA oligomers, RecA-covered oligonucleotides can be used for the development of the methods of the genome study or its modification, e.g. for long-distanced site-selective cleavage of DNA, arrest or activation transcription. However, these methods are not yet routine and more detailed examination of the site-selective interaction with DNA is needed.
The endonuclease R.EcoR124 will be purified and the target sides be cloned. The binding will be followed by gel retardation and EM experiments for different DNA/enzyme ratios. Furthermore DNA length measurements will be performed and curvator maps will be produced. The influence of supercoiled DNA will be investigated as well as the DNA/endonuclease complexes. PNA oligomers will be synthesized and clones will be made of the target sides.
This is the framework of a long-term study of the site-selective interaction of dsDNA with restriction-modification enzyme of the 1st type R.EcoR124 and RNA polymerase E.coli with PNA-induced loops.
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94805 Villejuif
France