Final Activity Report Summary - DNA ENZYMES (A multidisciplinary approach to the study of DNA enzymes down to the single molecule level)
Enzymes that interact with DNA are crucial for the copying, maintenance and repair, and expression of genetic information. Those DNA enzymes that interact with DNA at specific sites have to locate these sites, recognize the specific sequence and initiate catalysis. In the past, the focus of scientific investigation has been on the recognition process and the catalytic reaction, while the process of target site location on one hand and the conformational transitions required for coupling recognition and catalysis on the other hand, have received relatively little attention. The reasons for this are not hard to see: the processes proceeding and following recognition involve complexes, that are spatially and temporarily transient in nature, unsynchronised and often not highly populated. The study of such processes is experimentally very demanding; however, it is essential that an understanding of these processes is achieved before we can claim that we have an adequate description of the mode of action of these important enzymes.
There are many reasons why it is important to have a better understanding of DNA enzymes, among which are the following:
- They are essential tools for gene technology, and a thorough understanding of these systems will certainly enable better use to be made of them.
- Malfunction of several DNA enzymes, in particular DNA repair enzymes is causally related to human diseases, in particular some forms of cancer, in which human DNA repair proteins are affected; again, understanding their mechanism of action may eventually lead to new therapeutic concepts.
- A major goal in the post-genomic era is to develop tools for gene targeting which in principle could be used to repair defective genes, preferably via homologous recombination with DNA carrying an intact copy of the gene in question. Essential tools for this purpose are rare cutting restriction and homing endonucleases as well as other meganucleases. Their effective use for this purpose and their re-engineering requires a thorough understanding of their mechanism of action.
We set up an interdisciplinary and inter-sectorial network to achieve the following scientific goals:
- To understand the various mechanisms by which proteins or protein complexes locate their target sites on DNA.
- To understand how communication is achieved between recognition sites and sites of action, when these are separated by up to thousands of base pairs, and how ATP hydrolysis is used for this purpose.
- To understand how the catalytic event is triggered.
The nature of these scientific goals was such that the investigative approach to be taken had to be necessarily a multidisciplinary one, involving molecular biology, enzymology, bioorganic and biophysical chemistry, bioinformatics, as well as computational and structural biology.
Of special importance for the success of the project were recently developed single molecule techniques which allow studying the dynamics of unsynchronised processes in real time, even when the molecular species of interest is of low abundance.
We concentrated our studies on restriction endonucleases, DNA repair enzymes and DNA polymerases and made decisive progress in understanding the molecular basis of target site location and initiation of catalysis by these enzymes, and by extension, to DNA enzymes in general. Guided by bioinformatic analyses and structural studies these proteins were modified with fluorescent labels, such that their interaction with DNA could be followed in time and space with high resolution. This allowed understanding how individual proteins find their specific target sequence on DNA in vast excess of non-specific sequences and to carry out their catalytic reaction with high accuracy and speed.
There are many reasons why it is important to have a better understanding of DNA enzymes, among which are the following:
- They are essential tools for gene technology, and a thorough understanding of these systems will certainly enable better use to be made of them.
- Malfunction of several DNA enzymes, in particular DNA repair enzymes is causally related to human diseases, in particular some forms of cancer, in which human DNA repair proteins are affected; again, understanding their mechanism of action may eventually lead to new therapeutic concepts.
- A major goal in the post-genomic era is to develop tools for gene targeting which in principle could be used to repair defective genes, preferably via homologous recombination with DNA carrying an intact copy of the gene in question. Essential tools for this purpose are rare cutting restriction and homing endonucleases as well as other meganucleases. Their effective use for this purpose and their re-engineering requires a thorough understanding of their mechanism of action.
We set up an interdisciplinary and inter-sectorial network to achieve the following scientific goals:
- To understand the various mechanisms by which proteins or protein complexes locate their target sites on DNA.
- To understand how communication is achieved between recognition sites and sites of action, when these are separated by up to thousands of base pairs, and how ATP hydrolysis is used for this purpose.
- To understand how the catalytic event is triggered.
The nature of these scientific goals was such that the investigative approach to be taken had to be necessarily a multidisciplinary one, involving molecular biology, enzymology, bioorganic and biophysical chemistry, bioinformatics, as well as computational and structural biology.
Of special importance for the success of the project were recently developed single molecule techniques which allow studying the dynamics of unsynchronised processes in real time, even when the molecular species of interest is of low abundance.
We concentrated our studies on restriction endonucleases, DNA repair enzymes and DNA polymerases and made decisive progress in understanding the molecular basis of target site location and initiation of catalysis by these enzymes, and by extension, to DNA enzymes in general. Guided by bioinformatic analyses and structural studies these proteins were modified with fluorescent labels, such that their interaction with DNA could be followed in time and space with high resolution. This allowed understanding how individual proteins find their specific target sequence on DNA in vast excess of non-specific sequences and to carry out their catalytic reaction with high accuracy and speed.