This work will combine crystallographic and biochemical studies to understand the organization and regulation of a classic DNA transposase that is now a biotech tool, that of bacteriophage Mu. This is the most thoroughly characterized of the large and highly diverse """"""""DDE"""""""" family of transposases and retroviral integrases, and studies of Mu will continue to illuminate paradigms that translate to the rest of the family. Preliminary results include a recently-determined structure of an active Mu transposase - DNA complex, which contains 240 kDa of protein and 135bp of DNA. This is the largest and most complex structure of any transposase-DNA structure to date, and only the 2nd DDE recombinase to be crystallized with target DNA. This structure is serving as the springboard for further studies.
AIM 1 is to complete the current structure and to obtain the highest resolution crystal form possible. The current structure guided the construction of an active but more compact chimeric transposase with a different DNA binding domain that may aid crystallization, will facilitate in vitro experiments in our lab and others', and may aid in engineering the system to be more useful as a biotech tool.
AIM 2 is to understand the conformational changes that underlie increasing stability and ClpX recognition. Our structure suggests a model for how product binding energy is used by transposases to drive forward an otherwise isoenergetic chemical reaction, and how the extremely stable final complex can be preferentially recognized by the ATP-dependent unfoldase ClpX.
This aim uses bulk and single-molecule FRET and LRET, as well as some crystallography and solution assays.
AIM 3 is to understand the structural basis for assembly of with full left and right phage ends. Although an active complex requires only 4 copies of the transposase (which is what we have crystallized), the right and left ends of the phage contain different arrays of 3 transposase binding sites each. Our structure provides a basis for testable hypotheses regarding why the left end differs from the right and how it is incorporated into the final complex. This work will involve complex formation and activity assays with carefully chosen mutants specifically targeted to different binding sites. A detailed understanding of this system will provide an informative example for understanding why many other mobile DNA elements have different left and right ends, with seemingly-extra recombinase binding sites.

Public Health Relevance

Mobile genetic elements such as DNA transposons are widespread in nature, are useful biotechnology tools, and are also a common vector for the spread of antibiotic resistance. The mechanism used by many transposons to insert into the hosts'DNA is similar to that used by retroviruses such as HIV. The work proposed here will advance our basic understanding of the mechanism and regulation of DNA transposition.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-H (02))
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Janes, Daniel E
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University of Chicago
Schools of Medicine
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Rasila, Tiina S; Pulkkinen, Elsi; Kiljunen, Saija et al. (2018) Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering. Nucleic Acids Res 46:4649-4661
Fuller, James R; Rice, Phoebe A (2017) Target DNA bending by the Mu transpososome promotes careful transposition and prevents its reversal. Elife 6:
Toussaint, Ariane; Rice, Phoebe A (2017) Transposable phages, DNA reorganization and transfer. Curr Opin Microbiol 38:88-94
Arkhipova, Irina R; Rice, Phoebe A (2016) Mobile genetic elements: in silico, in vitro, in vivo. Mol Ecol 25:1027-31
Ling, Lorraine; MontaƱo, Sherwin P; Sauer, Robert T et al. (2015) Deciphering the Roles of Multicomponent Recognition Signals by the AAA+ Unfoldase ClpX. J Mol Biol 427:2966-82