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. 2017 Sep 19;45(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

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Free PMC article

Monitoring Replication Protein A (RPA) Dynamics in Homologous Recombination Through Site-Specific Incorporation of Non-Canonical Amino Acids

Nilisha Pokhrel et al. Nucleic Acids Res. .
Free PMC article

Abstract

An essential coordinator of all DNA metabolic processes is Replication Protein A (RPA). RPA orchestrates these processes by binding to single-stranded DNA (ssDNA) and interacting with several other DNA binding proteins. Determining the real-time kinetics of single players such as RPA in the presence of multiple DNA processors to better understand the associated mechanistic events is technically challenging. To overcome this hurdle, we utilized non-canonical amino acids and bio-orthogonal chemistry to site-specifically incorporate a chemical fluorophore onto a single subunit of heterotrimeric RPA. Upon binding to ssDNA, this fluorescent RPA (RPAf) generates a quantifiable change in fluorescence, thus serving as a reporter of its dynamics on DNA in the presence of multiple other DNA binding proteins. Using RPAf, we describe the kinetics of facilitated self-exchange and exchange by Rad51 and mediator proteins during various stages in homologous recombination. RPAf is widely applicable to investigate its mechanism of action in processes such as DNA replication, repair and telomere maintenance.

Figures

Figure 1.
Figure 1.
Position of non-canonical amino acid insertion in RPA. (A) Crystal structure of Ustilago maydis RPA bound to ssDNA is shown (PDB ID: 4GOP) with RPA70, RPA32 and RPA14 colored green, red and yellow, respectively. The zoomed-in image shows two loops, L-a and L-b, flanking the ssDNA (black sticks) and Trp-101 is shown as stick representation in blue. (B) Conservation of amino acid sequence in the region where Trp-101 resides in RPA32. W101 is highlighted in bold (red).
Figure 2.
Figure 2.
Insertion of ncaa and bio-orthogonal labeling of RPA. (A) Plasmids used for the overexpression of RPA and ncaa components. The three subunits of RPA are cloned into a pET vector and the RPA32 subunit is engineered to carry a C-terminal polyhistidine tag and a TAG inserted for the incorporation of 4AZP. The genes for the tRNA that recognizes the amber suppressor codon and inserts 4AZP (tRNACUA) and its corresponding tRNA synthetase are engineered into the pDULE2-pCNF plasmid. (B) SDS-PAGE analysis of RPAWT, RPA4ZAP and the MB543-labeled RPA (RPAf)proteins are shown after coommassie staining (left) or fluorescence imaging (right). Site-specific fluorescence labeling of the RPA32 subunit is observed.
Figure 3.
Figure 3.
ssDNA binding properties of RPAf. (A) Excitation and emission spectra of RPAf show maximum values at 555 nm (λex) and 566 nm (λem). (B) RPAf was excited at 535 nm and emission spectra were recorded in the absence or presence of ssDNA [(dT)97] orplasmid dsDNA. A ∼5% increase in fluorescence signal is observed when ssDNA is present in the reaction. (C) Electromobility band shift analysis (EMSA) of RPAWT (top) and RPAf (bottom) binding to 50 nM 32P-labeled (dT)35 oligonucleotides show bound and unbound complexes and (D) quantitation of the EMSA data show stoichiometric binding to ssDNA for both RPA and RPAf.
Figure 4.
Figure 4.
Kinetics of RPA binding to ssDNA. (A) Schematic of stopped-flow experiment to capture RPA–ssDNA binding kinetics. (B) A rapid change in RPAf fluorescence is observed upon binding to a (dT)97 ssDNA oligonucleotide (red trace), whereas no change in fluorescence is observed in the absence of DNA (black trace). (C) Fit of the stopped-flow data (dashed blue line) show the presence of a rapid (kobs,1 = 23 ± 1.2 s−1) and slow phase (kobs,2 = 0.003 ± 0.0006 s−1) for ssDNA dependent changes in RPAf fluorescence. Intrinsic tryptophan fluorescence changes in (D) RPAWT and (E) RPAf upon binding to ssDNA reveal rapid changes in fluorescence and fit of the data yield kobs,1 = 28 ± 1.8 s−1, kobs,2 = 0.014 ± 0.004 s−1 for RPAWT, and kobs,1 = 32 ± 3.8 s−1, kobs,2 = 0.008 ± 0.003 s−1 for RPAf, respectively. (F) Free Rad51 or Srs2 in the reaction do not affect the basal fluorescence of RPAf.
Figure 5.
Figure 5.
Facilitated exchange of RPAf on ssDNA by RPA and SSB. (A) Schematic of stopped-flow experiments to capture facilitated exchange of RPAf by unlabeled RPAWT. (B) Pre-formed RPAf-ssDNA complexes formed on (dT)97 ssDNA substrates are effectively displaced by unlabeled RPAWT. The rate of exchange increases with concentration of unlabeled RPAWT, and (C) yields an apparent observed rate of 0.7 ± 0.1 × 10−12 M−1 s−1 for facilitated exchange. (D) Schematic of facilitated exchange experiments with E. coli SSB, which (E) displaces or exchanges with RPAf more effectively than RPAWT with (F) an apparent observed rate of 47.3 ± 1.7 × 10−12 M−1 s−1.
Figure 6.
Figure 6.
Dynamics of RPAf during stages of homologous recombination. (A) Schematic of Rad51 binding to RPAf–ssDNA complexes. (B) Preformed RPAf–ssDNA complexes are not disrupted by Rad51 in the absence of ATP (gray trace), but effectively displaced in the presence of ATP (orange trace). The data is well described by a double exponential fit yielding kobs,1 = 0.26 ± 0.08 s−1 and kobs,2 = 0.02 ± 0.004 s−1. (C) Schematic of stopped-flow experiments to observe the effect of Srs2 on RPAf–ssDNA complex stability. The green arrow depicts potential rebinding of RPAf in the reaction. (D) Increasing concentrations of Srs2 show a small change in the fluorescence signal, but no significant change in overall fluorescence is observed. Insert shows an exponential phase with an observed rate constant of 8.5 ± 0.8 s−1. (E) Schematic of events during filament disassembly. The green arrows denote removal of RPAf upon Rad51 rebinding. (F) In filament clearing experiments, a preformed Rad51 filament prevents RPAf from binding to ssDNA (blue trace), however, when Srs2 is present in the reaction, a gradual increase in fluorescence is observed (pink trace) highlighting clearing of Rad51 molecules from ssDNA by Srs2 followed by RPAf binding to ssDNA. The data displays a single exponential profile (dotted line) with kobs = 0.005 ± 0.001 s−1.
Figure 7.
Figure 7.
ssDNA curtains to visualize RPAf. (A) Schematic of a RPA-coated double-tethered ssDNA curtain. (B) Representative examples of individual ssDNA molecules bound by RPAf (shown in magenta). The 5′-biotin tether is oriented at the top of each window that are all 2.7 × 13.5 μm. (C) Schematic showing the ssDNA curtain experiment time course beginning in the top panel with an RPA-coated ssDNA molecule. Injecting 2 μM Rad51 displaces RPAf from the ssDNA to form the pre-synaptic filament that is resistant to rebinding of RPAf in the middle panel. Flushing ATP from the system results in spontaneous Rad51 dissociation and the re-binding of RPAf. (D) A representative kymograph of a ssDNA molecule through time. At the start, ssDNA is already coated by RPAf and buffer containing 100 pM RPAf is flowing through the chamber at 0.2 ml/min. Switching to a buffer lacking RPAf shows that bound RPAf remains stable until the introduction of 2 μM wild type Rad51 when flow is stopped. The loss of RPAf signal is evidence that Rad51 outcompetes the bound RPAf to form a pre-synaptic filament. Resuming flow with buffer containing 100 pM RPAf and 2 mM ATP shows the ssDNA remains dark as RPAf cannot displace Rad51. However, switching to buffer with 100 pM RPAf and no ATP shows assembly and disassembly of a wild type Rad51 filament on an RPAf-coated ssDNA molecule.

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References

    1. Siebert G., Humphrey G.B. Enzymology of the nucleus. Adv. Enzymol. Related Areas Mol. Biol. 1965; 27:239–288. - PubMed
    1. Anderson B.J., Larkin C., Guja K., Schildbach J.F. Using fluorophore-labeled oligonucleotides to measure affinities of protein-DNA interactions. Methods Enzymol. 2008; 450:253–272. - PMC - PubMed
    1. Valuchova S., Fulnecek J., Petrov A.P., Tripsianes K., Riha K. A rapid method for detecting protein-nucleic acid interactions by protein induced fluorescence enhancement. Scientific Rep. 2016; 6:39653. - PMC - PubMed
    1. Song D., Graham T.G., Loparo J.J. A general approach to visualize protein binding and DNA conformation without protein labelling. Nat. Commun. 2016; 7:10976. - PMC - PubMed
    1. Hwang H., Myong S. Protein induced fluorescence enhancement (PIFE) for probing protein-nucleic acid interactions. Chem. Soc. Rev. 2014; 43:1221–1229. - PMC - PubMed

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