Andrzej Stasiak, MER
Andrzej Stasiak received his PhD in 1981 from the Institute of Biochemistry and Biophysics of Polish Academy of Sciences in Warsaw.
Keywords: Genetic recombination, DNA repair, DNA topology, DNA knots
Functional transitions of DNA structure
Our group is interested in the functional aspects of DNA structure and topology. More recently we become interested in the overall organization of genomes starting with arrangement of DNA in phage heads and ending with chromosome territories in higher eukaryotes. Some aspects of our interests go beyond biology, like for example statistical mechanics studies of knotted polymers. However, also these studies have biological or biophysical applications in helping to explain, for example, how DNA topoisomerases can efficiently distinguish knotted DNA molecules from unknotted ones. During recent years numerical methods used to model behavior of DNA molecules became the main method utilized by our group, although biochemistry and electron microscopy, in particular, remained essential part of our research.
ORGANIZATION OF NUCLEAR ARCHITECTURE
We are interested in revealing the underlying physical phenomena responsible for the overall organization of chromosomal territories in interphase nuclei. Using computer simulations we addressed the following three questions:
• Why are chromosomal territories with relatively high transcriptional activity usually closer to the center of nucleus than those
with lower activity?
• Why are actively transcribed genes usually located at the periphery of their chromosomal territories?
• Why are pair-wise contacts between active and inactive genes less frequent than those involving only active or only inactive genes?
Our simulation studies, suggest that transcription factories-mediated interchromosomal contacts are probably the main organizers of nuclear architecture in interphase cells.
Numerous experiments including single molecule manipulations studies revealed that type II DNA topoisomerases involved in the process of DNA replication show strong preference of action on DNADNA juxtapositions where the opposing duplexes wind around each other in a left-handed way. However, freshly replicated circular DNA molecules, such as bacterial chromosomes, prokaryotic and eukaryotic extrachromosomal elements and DNA viruses such as SV40, form DNA catenanes, in which the direction of winding of two sister molecules is right-handed. How then these type II DNA topoisomerases manage to be very efficient in the decatenation of freshly replicated DNA molecules? We have approached this problem using Monte Carlo and Brownian dynamics simulations. These simulations permitted us to reveal that multiply interlinked catenanes release
their mechanical constraint in form of a higher order coiling with left-handed DNA-DNA juxtapositions. This observation explains why the chirality bias of type II DNA topoisomerases that are known to be involved in DNA decatenation does not interfere with their action during initial stages of DNA decatenation.
ELECTRON MICROSCOPY STUDIES OF PROTEIN-DNA INTERACTIONS IMPLICATED IN THE PROCESS OF DNA RECOMBINATION
Our group has a long time experience in electron microscopy imaging of functional complexes formed with DNA by various proteins participating in the process of homologous recombination. Our studies contributed in a significant way to understanding the role and the mechanism of action of such proteins as RecA, RuvAB, RAD51, RAD52, DMC1, BRCA2 or FANCM. In a collaboration project involving group of Prof. M. Takahashi, we investigated inhibitory effects of peptides derived from BRCA2 protein on the action of RAD51 protein with the aim of finding biologically active peptides that could be used as inhibitors of DNA repair processes in cancer cells. In a collaboration project with Prof. JY Masson, we recently studied intereactions of PALB2 protein with DNA. This human protein plays an important control function in the process of recombinational DNA repair. This protein binds to DNA directly but also interacts with RAD51, BRCA2 and BRCA1 proteins. In vitro assays demonstrated that PALB2 protein stimulates RAD51-promoted homologous pairing. Using electron microscopy we visualized PALB2 binding to
recessed ends of duplex DNA. We hope that our studies will shed additional light on the process by which mutations in PALB2 and BRCA2 lead to development of cancers in affected patients.
CHROMATIN AS TOPO II INHIBITOR?
Theoretical predictions and our own computer simulations revealed that formation of chromosomal territories can proceed spontaneously when modeled chromatin fibers are not permitted to pass through each other. This has led us to propose that type II DNA topoisomerases are unable to perform passages between unperturbed chromatin fibers. Of course, type II DNA topoisomerases are essential for decatention of freshly replicated DNA and for the elimination of positive DNA supercoiling arising due to progressing strand separation. However this action may happen when the regular chromatin structure is not yet reestablished after DNA replication or when the chromatin is distorted by positive supercoiling that is known to induce dissociation of histones. Such distorted chromatin fibers with proteinfree DNA regions could of course serve for Topo II- mediated passages just like it is the case in standard in vitro reactions involving protein-free DNA. Our proposal that regular chromatin structure prevents topo II-mediated passages between chromatin fibers is mainly relevant during decondensation of chromosomes after mitosis and during establishing chromosome territories in interphase nucleus.
Representative recent publications
Gari, K., Décaillet, C., Stasiak, A. Z., Stasiak, A. & Constantinou, A. FANCM can promote branch migration of Holliday junctions and regression of replication forks. Molecular Cell, 29, 141-148, 2008.
Burnier, Y., Dorier, J. & Stasiak, A. DNA supercoiling inhibits DNA knotting. Nucleic Acids Research, 36, 4956-4963, 2008.
Demurtas, D., Amzallag, A., Rawdon, E.J., Maddocks, J.H., Dubochet, J. & Stasiak, A. Bending modes of DNA directly addressed by Cryo-Electron Microscopy of DNA minicircles. Nucleic Acids Research, 37, 2882-2893, 2009.
Dorier, J. & Stasiak, A. Topological origins of chromosomal territories. Nucleic Acids Research, 37: 6316 – 6322, 2009.
Mareduzzo, D.*, Orlandini, E., Stasiak, A.*, Sumners, D.W., Tubiana, L., & Micheletti, C. DNA-DNA interactions in bacteriophage capsids are responsible for the observed DNA knotting. PNAS, 106, 22269-22274, 2009. *Joint first authors
Hui, M. P., Galkin, V. E., Yu, X., Stasiak, A. Z., Stasiak, A., Waldor, M. K., & Egelman, E. H. ParA2, a Vibrio cholerae chromosome partitioning protein, forms left-handed helical filaments on DNA. PNAS, 107, 4590-4595, 2010.
Nommé, J., Renodon-Cornière, A., Asanomi, Y., Sakaguschi, K., Stasiak, A. Z., Stasiak, A.*, Norden, B., Tran, V. & Takahashi, M.*. Design of potent inhibitors of human Rad51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide. Journal of Medicinal Chemistry, 53, 5782-5791, 2010. * Joint corresponding authors.
Buisson, R., Dion-Côté, A.-M., Coulombe, Y., Launay, H., Cai, H., Stasiak, A. Z., Stasiak, A., Xia, B. & Masson, J.-M. Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination. Nature Structural & Molecular Biology, 17, 1247-1254, 2010.
Dorier, J. & Stasiak, A. The role of transcription factories-mediated interchromosomal contacts in the organization of nuclear architecture. Nucleic Acids Research, 38, 7410-7421, 2010.
Witz, G., Dietler, G. & Stasiak, A. Tightening of DNA knots by supercoiling facilitates their unknotting by type II DNA topoisomerases. PNAS, 108, 3608-3611, 2011.