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Monitoring Replication Protein a (RPA) Dynamics in Homologous Marquette University e-Publications@Marquette Biological Sciences Faculty Research and Biological Sciences, Department of Publications 9-19-2017 Monitoring Replication Protein A (RPA) Dynamics in Homologous Recombination Through Site-specific ncorI poration of Non- canonical Amino Acids Nilisha Pokhrel Marquette University Sofia Origanti Marquette University, [email protected] Eric Parker Davenport Marquette University Disha M. Gandhi Marquette University Kyle Kaniecki Columbia University See next page for additional authors Published version. Nucleic Acids Research, Vol. 45, No. 16 (September 19, 2017): 9413-9426. DOI. Authors Nilisha Pokhrel, Sofia Origanti, Eric Parker Davenport, Disha M. Gandhi, Kyle Kaniecki, Ryan A. Mehl, Eric C. Greene, Chris Dockendorff, and Edwin Antony This article is available at e-Publications@Marquette: https://epublications.marquette.edu/bio_fac/603 Published online 12 July 2017 Nucleic Acids Research, 2017, Vol. 45, No. 16 9413–9426 doi: 10.1093/nar/gkx598 Monitoring Replication Protein A (RPA) dynamics in homologous recombination through site-specific incorporation of non-canonical amino acids Nilisha Pokhrel1, Sofia Origanti1, Eric Parker Davenport1, Disha Gandhi2, Kyle Kaniecki3,4, Ryan A. Mehl5, Eric C. Greene3, Chris Dockendorff2 and Edwin Antony1,* 1Department of Biological Sciences, Marquette University, Milwaukee, WI 53201, USA, 2Department of Chemistry, Marquette University, Milwaukee, WI 53201, USA, 3Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA, 4Department of Genetics and Development, Columbia University, New York, NY 10032, USA and 5Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA Received May 10, 2017; Revised June 27, 2017; Editorial Decision June 29, 2017; Accepted July 09, 2017 ABSTRACT than one enzyme is present in the reaction, determining the sequence of binding, and how the presence of one en- An essential coordinator of all DNA metabolic pro- zyme influences another is required to determine the over- cesses is Replication Protein A (RPA). RPA orches- all mechanism of action. In multi-protein, steady-state ex- trates these processes by binding to single-stranded periments, the contribution of an individual enzyme is of- DNA (ssDNA) and interacting with several other DNA ten probed by varying the concentration of the target en- binding proteins. Determining the real-time kinetics zyme relative to all other components (including the DNA of single players such as RPA in the presence of mul- substrate). Such experiments do not yield the microscopic tiple DNA processors to better understand the asso- rate constants of the individual steps in the reaction which ciated mechanistic events is technically challenging. are required to decipher the complete mechanism of action. To overcome this hurdle, we utilized non-canonical Moreover, transient-kinetic tools are required to capture amino acids and bio-orthogonal chemistry to site- rapid conformational changes in proteins, and this infor- mation sheds light on how the various proteins interact with specifically incorporate a chemical fluorophore onto each other and the DNA template. The use of fluorescently- a single subunit of heterotrimeric RPA. Upon binding f labeled DNA substrates serve as excellent tools to moni- to ssDNA, this fluorescent RPA (RPA ) generates a tor overall reaction kinetics or to characterize the DNA quantifiable change in fluorescence, thus serving as binding/dissociation dynamics of a single protein (2–5). For a reporter of its dynamics on DNA in the presence of example, short oligonucleotides have been labeled with flu- multiple other DNA binding proteins. Using RPAf,we orophores to monitor the outcome of DNA recombina- describe the kinetics of facilitated self-exchange and tion (6), assembly/disassembly of Rad51 nucleoprotein fil- exchange by Rad51 and mediator proteins during aments (7,8), track protein movement on DNA and RNA various stages in homologous recombination. RPAf (9–11) and to capture DNA unwinding (12,13). Such an ap- is widely applicable to investigate its mechanism of proach is limited by the read out from the DNA substrate action in processes such as DNA replication, repair and the contribution of individual enzymes cannot be inter- preted when multiple proteins are present in a reaction. and telomere maintenance. To capture the sequence of binding and dynamics of each enzyme in a multi-protein reaction, one approach is to ob- INTRODUCTION tain a direct, quantifiable signal from a fluorescent label po- sitioned on the individual protein that can undergo a change Enzymes that bind and function on DNA are necessary for in fluorescence upon binding to DNA(9,14). The changes all DNA metabolic processes such as replication, recom- in fluorescence help ascertain enzyme dynamics onDNA bination, repair, and transcription. Multiple DNA bind- and how such dynamics are influenced by the presence of ing proteins function together to catalyze these processes other DNA binding proteins in the reaction. Ensemble and (1). Measuring the DNA binding properties of a single single-molecule based fluorescence spectroscopy serve as enzyme is relatively straightforward. However, when more *To whom correspondence should be addressed. Tel: +1 414 288 1474; Fax: +1 414 288 7357; Email: [email protected] Present address: Eric Parker Davenport, Applied Genetic Technologies Corporation (AGTC), Alachua, FL 32615, USA. C The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Downloaded from https://academic.oup.com/nar/article-abstract/45/16/9413/3956631 by Marquette University user on 06 June 2018 9414 Nucleic Acids Research, 2017, Vol. 45, No. 16 powerful tools to investigate enzyme function on nucleic (33,36,38). Defective HR leads to genomic rearrangements acid substrates, but are reliant on the generation of proteins and chromosomal defects often resulting in hereditary can- tagged with genetically encoded fluorophores such as eGFP cers and cancer-prone diseases such as Fanconi Anemia or mCherry (15–17). These tags are large in size, have posi- and Bloom’s syndrome (39,40). In addition to DNA repair, tional constraints, are likely to interfere with activity, (18) HR is fundamental to the maintenance of gametic diver- and potentially inhibit protein–protein interactions (19). sity during meiotic crossover events. During HR, dsDNA The attachment of large genetically-encoded fluorophores is at the site of a break is resected to yield long stretches of particularly problematic for smaller DNA binding proteins ssDNA, which serve as a template for the nucleation of such as Rad51, RecA or Dmc1, that function by forming co- the Rad51 recombinase and formation of nucleoprotein fil- operative nucleoprotein filaments on DNA. Finally, attach- aments. Rad51 functions as the central engine in HR by ment of genetically encoded fluorophores is often limited to performing ATP-dependent strand exchange (41,42). In the the N- and C-terminal ends of the candidate proteins. Due cell, the resected ssDNA is rapidly coated by RPA to pro- to the positional constraints, and their size, they cannot be tect it from nucleolytic degradation, and this step has been site-specifically introduced in internal regions of a protein, shown to activate the DNA damage response through sev- which is often necessary to capture conformational move- eral checkpoint kinases (36). To promote HR, RPA must ments and for Forster¨ Resonance Energy Transfer (FRET) first be displaced from the ssDNA, thus allowing formation studies (20). Small, chemical fluorophores circumvent many of the Rad51 nucleoprotein filament. Pro-recombinogenic of these issues, and can be site-specifically placed anywhere mediator proteins such as Rad52 and BRCA2 have been in the primary sequence of a protein. shown to promote Rad51 binding to ssDNA by displac- The most commonly used approach to site-specifically ing RPA (43–45). The Rad51 nucleoprotein filament then attach fluorescent probes on proteins relies on maleimide- catalyzes strand exchange to drive HR. Similarly, in other thiol chemistry to cysteine residues (21). While robust, this instances where HR is inhibited, Rad51 filaments are dis- approach suffers from the need to generate a single-cysteine assembled by anti-recombinogenic mediators such as the bearing functional version of the target protein. This ap- Srs2 helicase (7,46,47). In Rad51 filament clearing reac- proach is not feasible in proteins where multiple cysteines tions, RPA is proposed to sequester naked ssDNA once the are important for function or folding. In addition, screen- Rad51 molecules are removed by Srs2 (48). While RPA has ing and isolating modifiable cysteines in multi-cysteine pro- been shown to regulate several steps in HR, its precise mode teins is time-intensive. This problem is circumvented using of action in these events is not clearly understood. non-canonical amino acids (ncaa), an attractive strategy to The complexity of RPA function in multi-protein con- engineer site-specific fluorescently labeled versions of pro- texts necessitates the need for a site-specifically
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