The four-way DNA junction known as the Holliday junction (HJ) figures prominently in DNA recombination, replication and repair;it is the central intermediate in both homologous and site-specific recombination, where genetic, biochemical and structural studies of the HJ have been particularly fruitful. We have been studying formation, properties, and resolution of this recombination intermediate in the ? phage recombination pathway, a paradigm for a large family of site-specific recombinases that administers a wide range of functions in prokaryotes, eukaryotes, and archaea. Recombinases of this family catalyze rearrangements between DNA sequences (called att sites in the ? system) with very little homology to each other and have the ability to generate and resolve HJs without the input of energy. Experiments in this proposal grow out of a large body of biochemical, genetic, and structural data that afford insights into how HJs are generated and resolved by the ? phage-encoded Int protein, which is also a model for the large subset of virally-encoded family members that are heterobivalent DNA binding proteins. The four specific aims address questions growing out of results obtained during the previous project period and/or of long standing in the field. They are: 1) to test the functional implications of the crystal structure of HJ complexed with Int and arm-type oligonucleotides (HJ-Int-Arm complex);2) to determine which chemical and/or conformational steps are required to form a stable synaptic complex;3) to determine the rate limiting step in formation of the HJ;4) to determine the numerical distribution of attL-bound and attR-bound Integrase subunits that lead to successful synaptic events and the subsequent formation of stable Holliday junction.
Aims 1 -3 depend upon approaches developed and utilized during the previous project period.
Aim 4 involves a logical extension of some of those techniques. Several of the questions posed in aims 2-4 would be extremely difficult to answer using ensemble biochemistry since many of the relevant intermediates are transient and/or difficult to verify as being on-pathway events. We propose to address these questions using logical extensions of our investment in single molecule approaches to studying site-specific recombination (something which is thus far unique in the field). The four-way DNA junction known as the Holliday junction figures prominently in DNA recombination, replication and repair. We have been studying formation and resolution of this recombination intermediate in the ? phage-encoded recombination pathway, a paradigm for a large family of site-specific recombinases that is ubiquitous in nature and administers a wide range of functions, many of which figure prominently in various aspects of health-related issues.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Portnoy, Matthew
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Brown University
Schools of Medicine
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Laxmikanthan, Gurunathan; Xu, Chen; Brilot, Axel F et al. (2016) Structure of a Holliday junction complex reveals mechanisms governing a highly regulated DNA transaction. Elife 5:
Landy, Arthur (2015) The ? Integrase Site-specific Recombination Pathway. Microbiol Spectr 3:MDNA3-0051-2014
Tong, Wenjun; Warren, David; Seah, Nicole E et al. (2014) Mapping the ? Integrase bridges in the nucleoprotein Holliday junction intermediates of viral integrative and excisive recombination. Proc Natl Acad Sci U S A 111:12366-71
Seah, Nicole E; Warren, David; Tong, Wenjun et al. (2014) Nucleoprotein architectures regulating the directionality of viral integration and excision. Proc Natl Acad Sci U S A 111:12372-7
Matovina, Mihaela; Seah, Nicole; Hamilton, Theron et al. (2010) Stoichiometric incorporation of base substitutions at specific sites in supercoiled DNA and supercoiled recombination intermediates. Nucleic Acids Res 38:e175
Warren, David; Laxmikanthan, Gurunathan; Landy, Arthur (2008) A chimeric Cre recombinase with regulated directionality. Proc Natl Acad Sci U S A 105:18278-83
Hazelbaker, Dane; Azaro, Marco A; Landy, Arthur (2008) A biotin interference assay highlights two different asymmetric interaction profiles for lambda integrase arm-type binding sites in integrative versus excisive recombination. J Biol Chem 283:12402-14
Sun, Xingmin; Mierke, Dale F; Biswas, Tapan et al. (2006) Architecture of the 99 bp DNA-six-protein regulatory complex of the lambda att site. Mol Cell 24:569-80
Mumm, Jeffrey P; Landy, Arthur; Gelles, Jeff (2006) Viewing single lambda site-specific recombination events from start to finish. EMBO J 25:4586-95
Radman-Livaja, Marta; Biswas, Tapan; Ellenberger, Tom et al. (2006) DNA arms do the legwork to ensure the directionality of lambda site-specific recombination. Curr Opin Struct Biol 16:42-50

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