Intellectual Merit. Genomes undergo several types of rearrangements as part of normal cell growth, development and differentiation. Such DNA rearrangements fall into several categories, including homologous recombination, site-specific recombination, DNA transposition, and retro-transposition. These rearrangements proceed in a highly orderly and regulated manner instituted by high-order interactions of proteins with cognate DNA sequences as well as interactions among the proteins themselves. Proper action of site-specific recombinases requires proper geometry of the DNA dictated by two features: a) chirality, the handedness (right or left) in which DNA sites come together during a particular reaction, and b) alignment of the DNA molecules at the recombination site (parallel vs. anti-parallel). This project will use difference topology analysis to address two major questions about the mechanism of action of tyrosine family recombinases. Is there a defined chirality to the interaction of target sites? And how does the DNA sequence of recombination sites affect alignment of the target sites? The results of these studies will yield important insights with potential applications to genome engineering and biotechnology.
Broader Impacts. The project will provide interdisciplinary training opportunities at the interface of mathematics and biology for a postdoctoral fellow, a graduate student and undergraduates. In addition, this interdisciplinary focus will be featured in classroom activities for graduate and undergraduate courses and in presentations for the lay public.
Normal 0 false false false EN-US X-NONE X-NONE Fields of study: Site-specific DNA recombinases mediate precise DNA rearrangements with important regulatory and developmental consequences. We investigate the mechanism of action of the tyrosine family recombinase Flp, which plays a central role in controlling the copy number of a selfish plasmid present in Saccharomyces yeast. We also study the mechanisms by which the plasmid propagates itself with nearly chromosome-like stability in host cell populations. Accomplishments: In the present study, we have characterized the topological and geometric features of Flp mediated recombination between its target DNA sites, FRTs. Our findings suggest that the FRT sites are arranged in a specific geometry (anti-parallel) within the recombination complex. When the FRT partner sites are incompatible because of sequence differences between their exchanged DNA segments, the geometry for site alignment is not altered. Furthermore, the DNA exchange reaction is not blocked. The first recombination event between the incompatible partners results in products containing non-complementary base pairs. They are unstable, and undergo a second recombination event after the initial complex disassembles and a new recombination complex is assembled. The net effect is that recombination is annulled; however, the two sequential recombination events can tie supercoiled substrate molecules into knots. These conclusions were arrived at from experiments utilizing topological analyses of DNA by biochemical and electron microscopy assays. They were also confirmed by collaborative experiments based on fluorescence resonance energy transfer properties of single DNA molecules. In ongoing experiments, we are testing the notion that recombinase enzymes such as Flp introduce crossings of a specific handedness when they interact with their target sites. We deduce the sign of such crossings (right or left handed) from the topological characterization of the recombination products: knotted DNA circles as well as interlinked DNA circles. Related accomplishments: With permission from NSF, we also investigated another DNA topology-related problem in the stable propagation of the yeast selfish plasmid. We demonstrated that the plasmid partitioning locus has a non-standard positive supercoil analogous to that of the point centromere of yeast chromosomes. This topological similarity is consistent with a number of circumstantial pieces of evidence suggesting that the atypical point centromere originated from the partitioning locus of an ancestral plasmid. Our initial findings have been published in two papers, one in Molecular and Cellular Biology, and the other in the Proceedings of the National Academy of Sciences (USA). The potential evolutionary relationship between the point centromere and the plasmid partitioning locus is an active area of our research interest. Inter-disciplinary approaches; training of students and post-doctoral fellows; teaching: The completed and ongoing investigations encompass the fields of mathematics, biochemistry and biophysics. One graduate student and one post-doctoral fellow, with assistance from a research scientist associate, were involved in the topological studies of recombination. Additionally, two undergraduate students performed recombination-related experiments as part of their research training. A graduate student and a post-doctoral fellow contributed to the collaborative biophysical investigations on the assembly of the recombination complex. The general topological principles that form the underpinnings of site-specific recombination were covered in four lectures of an upper division undergraduate course. Publications: The completed studies were recently published in the Journal of Molecular Biology. A commentary on the work from other groups pertaining to the topology of DNA replication was published in the Proceedings of the National Academy of Sciences (USA). Normal 0 false false false EN-US X-NONE X-NONE
