Prof. Dr. Alfred Pingoud: Facilitated diffusion to locate specific target site on the DNA
State-of-the-art and preparatory work
Enzymes that interact with DNA are crucial for the copying, maintenance and repair, and expression of genetic information. Those enzymes that interact with DNA at specific sites have to locate these sites, recognize the specific sequence and initiate catalysis.
Among these enzymes, type II restriction endonucleases are the most intensively studied enzymes (1). In the past, the focus of scientific investigation has been on the recognition process and the catalytic reaction of restriction enzymes, while the process of target site location and the conformational transitions required for coupling recognition and catalysis have received relatively little attention (1). Highly sophisticated fluorescence techniques, especially recently developed single molecule techniques, allow addressing these questions (2).
It is widely accepted that, in order to reach their target site, proteins first translocate along nonspecific DNA. The mechanism underlying this so‐called ‘facilitated diffusion’, however, is still under debate (3). For EcoRV we have reported recently the first direct observation of “sliding” and “jumping” of individual molecules on nonspecific DNA (4, 5). The contribution of sliding/jumping to facilitated diffusion, however, might differ from protein to protein, depending on both the structure of the apoenzyme and its complexes with non‐specific and specific DNA and the dynamics of complex formation (6). Another open question is whether the sliding of the enzymes along the DNA double strand follows the pitch of the double helix or not (7). The approach that will be taken will allow differentiating between a sliding or “hopping” mechanism, which is a controversial subject discussed in the literature. The results that we hope to obtain and the methods to be developed will be of general importance as presumably all proteins that recognize particular features on the DNA (sequences, modifications, damage) will have to make use of some form of facilitated diffusion.
1. Pingoud, A., Fuxreiter, M., Pingoud, V. & Wende, W. (2005) Type II restriction endonucleases: structure and mechanism. Cell Mol Life Sci 62, 685‐707.
2. Gorman, J. & Greene, E. C. (2008) Visualizing one‐dimensional diffusion of proteins along DNA. Nat Struct Mol Biol 15, 768‐774.
3. Halford, S. E. (2009) An end to 40 years of mistakes in DNA‐protein association kinetics? Biochem Soc Trans 37, 343‐348 4. Bonnet, I., Biebricher, A., Porte, P.L., Loverdo, C., Benichou, O., Voituriez, R., Escude, C., Wende, W., Pingoud, A. & Desbiolles, P. (2008) Sliding and jumping of single EcoRV restriction enzymes on non‐cognate DNA. Nucleic Acids Res 36, 4118‐4127.
5. Biebricher, A., Wende, W., Escude, C., Pingoud, A. & Desbiolles, P. (2009) Tracking of single quantum dot labeled EcoRV sliding along DNA manipulated by double optical tweezers. Biophys J 96, L50‐52.
6. Pingoud, A. & Wende, W. (2007) A sliding restriction enzyme pauses. Structure 15, 391‐393.
7. Blainey, P.C., Luo, G., Kou, S.C., Mangel, W.F., Verdine, G.L., Bagchi, B. & Xie, X.S. (2009) Nonspecifically bound proteins spin while diffusing along DNA. Nat Struct Mol Biol 16, 1224‐1229.
The overall aims of this project are
I. to elucidate whether type II restriction endonucleases “rotate” to maintain a specific orientation with respect to the DNA helix while diffusing along DNA.
II. to investigate the contribution of sliding/jumping to facilitated diffusion for structurally different type II restriction endonucleases and homing endonucleases.
III. to compare the process of target site location for restriction enzymes and their companion modification enzymes (DNA methyltransferases), e.g., R.EcoRV and M.EcoRV.
Work programme and methods
We intend to study the target site location of selected enzymes which recognize specific DNA target sequences. To this aim we produce specific fluorescently labelled enzymes which can be directly observed interacting with elongated DNA molecules using Total Internal Reflection Fluorescence Microscopy (TIRFM) in collaboration with P. Desbiolles (Paris). We could recently show that our setup works successfully for the type II restriction endonuclease EcoRV. In order to expand our study to restriction enzymes with different structural features we selected PvuII, the smallest restriction enzyme with a more “open” protein ‐ DNA interface, and BsoBI which fully encircles the DNA forming a tunnel. We will extend these studies to modification enzymes (M.EcoRV) and the homing endonucleases (e.g. I‐SceI, PI‐SceI) which recognize specific sequences up to 40 bp in length. For all proteins, single cysteine variants have to be produced (already done for PI‐SceI) which can be specifically modified by appropriate fluorophores or quantum dots. Biochemical characterization of the modified proteins must ensure that the enzymes are not impaired in binding and cleaving (modifying) the DNA. The results will show the impact of the different structural architectures on facilitated diffusion and the enzymatic process.
In order to address the question whether the proteins follow the helical pitch of the DNA while sliding along DNA, we intend to produce fusions of selected homodimeric restriction enzymes with extended quasi‐linear protein or DNA structures which are fluorescently labelled at their distal ends. This should allow following the movement of the widely separated fluorescent labels by TIRFM with superior spatial resolution and thereby allow deciding whether restriction enzymes, modification enzymes and homing endonucleases follow the helical geometry of the DNA. To this end we will fuse selected restriction enzymes with an engineered Rop protein which forms a stable extended coiled coil (M. Kokkinidis, Heraklion), or alternatively, with a double stranded DNA or PNA. The proteinprotein and protein‐nucleic acid fusions will be fluorescently labelled using appropriate fluorophores. The labelled constructs will be characterized extensively by biochemical and biophysical techniques before they can be studied in their interaction with DNA using single molecule techniques.
Titles for dissertations (prospective)
• Analysis of the facilitated diffusion of restriction enzymes and their accompanying methyltransferases
• Analysis of the sliding mechanism of restriction enzymes using protein‐protein and protein‐nucleic acid fusions
Relationships/connections within the research training group
Oretskaya, Kubareva: Synthesis of bifunctional crosslinkers and modified oligodeoxyribonucleotides
Aleksevskiy/Spirin/Karyagina: Molecular modelling of protein – protein conjugates
Benefits of the scientific exchange
The early stage and more experienced researchers (doctoral students and postdocs, respectively) involved in the project will get acquainted with molecular modelling and bioorganic chemistry and learn how the interdisciplinary cooperation between biochemists, chemists and physicists will benefit the progress of the project.