Molecular flexibility of DNA as a key determinant of RAD 51 recruitment
2020; Springer Nature; Volume: 39; Issue: 7 Linguagem: Inglês
10.15252/embj.2019103002
ISSN1460-2075
AutoresFederico Paoletti, Afaf H. El‐Sagheer, Jun Allard, Tom Brown, Omer Dushek, Fumiko Esashi,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle14 January 2020Open Access Source DataTransparent process Molecular flexibility of DNA as a key determinant of RAD51 recruitment Federico Paoletti Federico Paoletti Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Afaf H El-Sagheer Afaf H El-Sagheer Department of Chemistry, University of Oxford, Oxford, UK Department of Science and Mathematics, Suez University, Suez, Egypt Search for more papers by this author Jun Allard Jun Allard Department of Mathematics, University of California, Irvine, CA, USA Search for more papers by this author Tom Brown Tom Brown Department of Chemistry, University of Oxford, Oxford, UK Search for more papers by this author Omer Dushek Corresponding Author Omer Dushek [email protected] orcid.org/0000-0001-5847-5226 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Fumiko Esashi Corresponding Author Fumiko Esashi [email protected] orcid.org/0000-0003-0902-2364 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Federico Paoletti Federico Paoletti Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Afaf H El-Sagheer Afaf H El-Sagheer Department of Chemistry, University of Oxford, Oxford, UK Department of Science and Mathematics, Suez University, Suez, Egypt Search for more papers by this author Jun Allard Jun Allard Department of Mathematics, University of California, Irvine, CA, USA Search for more papers by this author Tom Brown Tom Brown Department of Chemistry, University of Oxford, Oxford, UK Search for more papers by this author Omer Dushek Corresponding Author Omer Dushek [email protected] orcid.org/0000-0001-5847-5226 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Fumiko Esashi Corresponding Author Fumiko Esashi [email protected] orcid.org/0000-0003-0902-2364 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Author Information Federico Paoletti1, Afaf H El-Sagheer2,3, Jun Allard4, Tom Brown2, Omer Dushek *,1,‡ and Fumiko Esashi *,1,‡ 1Sir William Dunn School of Pathology, University of Oxford, Oxford, UK 2Department of Chemistry, University of Oxford, Oxford, UK 3Department of Science and Mathematics, Suez University, Suez, Egypt 4Department of Mathematics, University of California, Irvine, CA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +44 1865 275576; E-mail: [email protected] *Corresponding author. Tel: +44 1865 275289; E-mail: [email protected] The EMBO Journal (2020)39:e103002https://doi.org/10.15252/embj.2019103002 See also: S Subramanyam & M Spies (April 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The timely activation of homologous recombination is essential for the maintenance of genome stability, in which the RAD51 recombinase plays a central role. Biochemically, human RAD51 polymerises faster on single-stranded DNA (ssDNA) compared to double-stranded DNA (dsDNA), raising a key conceptual question: how does it discriminate between them? In this study, we tackled this problem by systematically assessing RAD51 binding kinetics on ssDNA and dsDNA differing in length and flexibility using surface plasmon resonance. By directly fitting a mechanistic model to our experimental data, we demonstrate that the RAD51 polymerisation rate positively correlates with the flexibility of DNA. Once the RAD51-DNA complex is formed, however, RAD51 remains stably bound independent of DNA flexibility, but rapidly dissociates from flexible DNA when RAD51 self-association is perturbed. This model presents a new general framework suggesting that the flexibility of DNA, which may increase locally as a result of DNA damage, plays an important role in rapidly recruiting repair factors that multimerise at sites of DNA damage. Synopsis Kinetic studies of human RAD51 binding to DNA combined with mathematical modelling reveal that RAD51 preferentially polymerises on flexible DNA, explaining how RAD51 is recruited favourably to broken DNA and why certain rigid genomic regions, such as poly(dA) stretches, are left unrepaired. RAD51 polymerisation on ssDNA and dsDNA requires a minimal nucleus of four and two molecules, respectively. RAD51 polymerises faster on flexible ssDNA compared to rigid ssDNA, such as poly(dA), and dsDNA. RAD51 polymers, once assembled on DNA, stably associate with DNA independently of its flexibility. RAD51 polymers dissociate rapidly from flexible DNA, but not from rigid DNA, when RAD51 protomer-protomer interaction is perturbed. Introduction DNA double-strand breaks (DSBs) are cytotoxic lesions that can lead to chromosomal breaks, genomic instability and tumorigenesis in mammalian cells (Tubbs & Nussenzweig, 2017). Homologous recombination (HR) can offer an error-free DNA repair mechanism to restore genetic information at DSB sites and, in this way, contribute to genome stability. During HR-mediated repair, single-stranded DNA (ssDNA) overhangs are generated and rapidly coated with the ssDNA-binding replication protein A (RPA) (Chen & Wold, 2014). ssDNA-bound RPA is then exchanged for RAD51, the central ATP-dependent recombinase that catalyses HR-mediated repair. RAD51 polymerises on ssDNA to form a nucleoprotein filament and guide homologous strand invasion and DSB repair (Baumann et al, 1996). The central mechanism of HR is evolutionarily highly conserved, and the bacterial RAD51 ortholog RecA has clear preference to polymerise on ssDNA over dsDNA (Benson et al, 1994). However, human RAD51 shows weaker binding affinity to ssDNA compared to RecA, and the mechanism by which RAD51 polymerises on ssDNA in preference to dsDNA remains enigmatic. Earlier studies using electrophoretic mobility shift assay (EMSA) have suggested that RAD51 binds both ssDNA and dsDNA with similar affinities (Benson et al, 1994), albeit its preferential ssDNA binding was visible in the presence of ammonium sulphate (Shim et al, 2006). These endpoint assays, however, do not provide information as to whether binding kinetics may contribute to a potential RAD51-dependent ssDNA/dsDNA discrimination mechanism. Indeed, more recent kinetic studies have revealed that RAD51 polymerisation consists of two phases: a rate-limiting nucleation phase and a growth phase (Miné et al, 2007; Van der Heijden et al, 2007; Hilario et al, 2009). A minimal polymer nucleus, with a length of either two-to-three or four-to-five RAD51 protomers (Van der Heijden et al, 2007; Hilario et al, 2009; Subramanyam et al, 2016), is proposed to elicit the growth phase of RAD51 polymerisation. Intriguingly, RAD51 was shown to display faster association kinetics on ssDNA (Candelli et al, 2014) and slower dissociation kinetics on dsDNA, indicating that the RAD51-dsDNA complex is stable once formed (Miné et al, 2007). However, the mechanism underlying these kinetic differences is ill-defined. A key difference between ssDNA and dsDNA is their molecular flexibility: ssDNA is known to be more flexible compared to dsDNA. The flexibility of a DNA molecule can be characterised by its persistence length (Lp), a mechanical parameter quantifying polymer rigidity: the higher the persistence length, the more rigid the polymer. In the presence of monovalent or divalent salt, dsDNA displays an Lp of ~ 30–55 nm (Baumann et al, 1997; Brunet et al, 2015), while ssDNA is much more flexible, with an Lp of 1.5–3 nm (Murphy et al, 2004; Chi et al, 2013; Kang et al, 2014). These observations imply that ssDNA can explore a much larger configurational space compared to dsDNA. It follows that the formation of a structured (less flexible) RAD51 polymer on ssDNA will need to offset a greater deal of ssDNA's configurational freedom, referred to as entropic energy, compared to the formation of a RAD51 polymer on dsDNA. Despite the clear thermodynamic implications of RAD51 polymerisation on DNA, the impact of DNA flexibility on RAD51 nucleoprotein filament formation has been largely overlooked. In this study, we describe how RAD51 polymerises on DNA. Using a combination of surface plasmon resonance (SPR) and small-angle X-ray scattering (SAXS), we have assessed the RAD51 binding kinetics on DNA using ssDNA and dsDNA oligonucleotides differing in length and flexibility. Analyses of the SPR data using biochemical mathematical models revealed that RAD51 polymerisation required a minimal nucleus of four and two molecules on ssDNA and dsDNA, respectively. Interestingly, our analyses further uncovered that RAD51 is a mechano-sensor, a biomolecule that polymerises faster on more flexible DNA. This is surprising, because polymerisation on more flexible DNA should produce less stable polymers due to a larger configurational confinement. Therefore, we hypothesised that this confinement cost, defined as entropic penalty, is offset by a strong RAD51 protomer–protomer interaction and show this to be the case by analysing a RAD51 point mutant which is defective in self-association. We propose that RAD51 sensitively recognises locally flexible DNA, which may be generated at sites of DNA damage, and so rapidly forms nucleoprotein filament for HR repair. Results RAD51 preferential binding to ssDNA is dependent on the length of the DNA template To evaluate how human RAD51 discriminates between ssDNA and dsDNA, we used SPR to assess the binding kinetics of untagged recombinant human wild-type (WT) RAD51 to a 50-mer mixed-base ssDNA molecule (dN-50) and a 50-mer mixed-base paired dsDNA molecule (dN-50p) (Table 1). These analyses, conducted in the presence of ATP and Ca2+ to block RAD51 ATP hydrolysis (Bugreev & Mazin, 2004), showed (i) faster RAD51 association with ssDNA compared to dsDNA, and (ii) similar RAD51 lifetimes on both ssDNA and dsDNA (Fig 1A and B). These observations confirm previous findings that WT RAD51 distinguishes ssDNA from dsDNA through faster polymerisation, but not the stability of polymers, on ssDNA (Candelli et al, 2014), validating SPR as a sensitive experimental system with which to determine the kinetics of RAD51 binding to DNA. Table 1. The list of DNA oligonucleotides used in this study Name Mass (kDa) Sequence ssDNA dN-5 2.057 5′-CGGAC-Biotin TEG-3′ dsDNA dN-5p 4.286 5′-CGGAC LL GTCCG-Biotin TEG-3′ ssDNA dN-8 2.594 5′-CTGACTGC- Biotin TEG-3′ dsDNA dN-8p 6.139 5′-CTGACTGC LL GCAGTCAG-Biotin TEG-3′ ssDNA dN-11 3.926 5′-CGTCGATAGGC-Biotin TEG-3′ dsDNA dN-11p 7.993 5′-CGTCGATAGGC LL GCCTATCGACG-Biotin TEG-3′ ssDNA dN-14 4.872 5′-CGTCGATGAGCAGT-Biotin TEG-3′ ssDNA dN-17 5.795 5′-CGTCGATGAGCAGTGTC-Biotin TEG-3′ ssDNA dN-50 15.682 5′-Biotin-TTGAGAGAGCAGACCACAATTATCACCTACACGACATCATTTTATATCAA-3′ dsDNA dN-50p 31.157 (Forward) 5′-Biotin-TCGAGAGGGTAAACCACAATTATCGCCTACCCAAAACTATTTTATATCAA-3′ (Reverse) 5′-TTGATATAAAATAGTTTTGGGTAGGCGATAATTGTGGTTTACCCTCTCGA-3′ ssDNA dT-50 15.541 5′-Biotin-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3′ ssDNA dA-50 15.992 5′-Biotin-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3′ Biotin TEG: biotin linked to a flexible triethylene glycol spacer; Biotin: biotin linked to a standard C6 spacer; dA: deoxyadenosine; dN: mixed-base composition; dT: deoxythymidine; L: hexaethylene glycol flexible linker. Figure 1. The experimental setup A. Depiction of the experimental setting. A biotinylated DNA oligonucleotide is immobilised onto a surface plasmon resonance (SPR) CM5 chip via biotin–streptavidin interaction. RAD51 protein is injected over the DNA-coated SPR matrix to measure polymerisation kinetics. Throughout this period, association and dissociation of RAD51 take place simultaneously. Following protein injection (stop), running buffer is injected to measure dissociation kinetics. B. Wild-type (WT) RAD51 SPR curves for ssDNA dN-50 and dsDNA dN-50p. RAD51 was injected at 150 nM in the presence of 2.5 mM ATP (pH 7.5) and 10 mM CaCl2. The normalised mean number of WT RAD51 bound to respective DNA oligonucleotides (N) is plotted versus time, as measured by SPR, following the equation N = S/(L * (MRAD51/MDNA)), where S is the signal in RU units (1 RU ˜ 50 pg/mm2), L is the amount of DNA ligand immobilised onto the experimental flow cell (RU), MRAD51 is the molecular weight (kDa) of RAD51 (˜ 37 kDa) and MDNA is the molecular weight (kDa) of the immobilised DNA molecule. Source data are available online for this figure. Download figure Download PowerPoint We then aimed to define the initial steps of RAD51 association with ssDNA. To this end, we generated a series of mixed-base short ssDNA oligonucleotides with varying lengths, each of which can be bound by a restricted number of RAD51 molecules. As a single RAD51 molecule engages with three nucleotides of DNA (Short et al, 2016), a ssDNA consisting of five nucleotides (dN-5), eight nucleotides (dN-8), 11 nucleotides (dN-11), 14 nucleotides (dN-14) and 17 nucleotides (dN-17) can accommodate up to one, two, three, four and five RAD51 molecules, respectively (Fig 2A and Table 1). Our systematic SPR measurements of WT RAD51 binding kinetics revealed no RAD51 binding to the dN-5, dN-8 and dN-11, slow association and moderate dissociation with dN-14, and slightly faster association and slow dissociation with dN-17 (Fig 2A and B). These observations suggested that three or fewer RAD51 molecules are unable to generate a stable nucleus on ssDNA, four RAD51 molecules form a quasi-stable nucleus, and five or more RAD51 molecules are able to form a highly stable nucleus. Figure 2. Kinetics of WT RAD51 binding to ssDNA and dsDNA oligonucleotides of varying length A. Biotinylated ssDNA oligonucleotides of indicated lengths were separately immobilised onto SPR CM5 chips via biotin–streptavidin interaction. Wild-type (WT) RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics. B. The dotted and solid curves show the normalised mean number of RAD51 bound to the ssDNA oligonucleotides, as measured by SPR (see Fig 1 for equation) and the ODE model fits (see Fig 3A for model description), respectively. C. As in (A), except biotinylated dsDNA molecules of indicated lengths were used to measure association and dissociation kinetics. D. As in (B), the dotted and solid curves show the mean number of RAD51 bound to the dsDNA oligonucleotides, as measured by SPR and the ODE model fits (see Fig 3B for model description), respectively. Source data are available online for this figure. Download figure Download PowerPoint To similarly evaluate the initial steps of RAD51 association with dsDNA, we generated an analogous series of mixed-base dsDNA consists of five base pairs (dN-5p), eight base pairs (dN-8p) and 11 base pairs (dN-11p), which can accommodate up to one, two or three RAD51 molecules, respectively (Fig 2C and Table 1). Our SPR measurements detected no RAD51 binding to the dsDNA dN-5p, as was the case for the ssDNA dN-5. To our surprise, however, we detected slow association and moderate dissociation of WT RAD51 with the dN-8p, and even faster association and slower dissociation with the dN-11p (Fig 2D). These observations suggested that, while RAD51 molecule is unable to associate stably with dsDNA, two RAD51 molecules form a temporary stable (quasi-stable) nucleus and three or more RAD51 molecules are able to form a highly stable nucleus. This suggested that WT RAD51 nucleation on dsDNA requires only two-to-three RAD51 molecules versus four-to-five on ssDNA. Altogether, these observations support the idea that WT RAD51 can bind more strongly to short (≤ 11 bp) dsDNA compared to short (≤ 11 nt) ssDNA molecules, but binds more strongly to long (50 nt) ssDNA molecules compared to long (50 bp) dsDNA molecules. RAD51 polymerisation on ssDNA is promoted by faster adsorption and/or elongation compared to dsDNA Informed by these SPR datasets, we developed an ordinary differential equation (ODE) model with three modules: RAD51 polymerisation in solution, RAD51 polymerisation on ssDNA and RAD51 polymerisation on dsDNA. This model was globally fitted to all corresponding SPR curves in order to determine the mechanistic differences in RAD51 polymerisation kinetics on ssDNA and dsDNA. The module of RAD51 polymers in solution was modelled on the basis of mass action kinetics with a maximum length of 16 (see Appendix Methods and Appendix Table S1 for details). The model allows any RAD51 n-mer (1 ≤ n < 16) to associate with any other RAD51 m-mer (1 ≤ m < 16) to form a (m + n)-mer (1 < m + n ≤ 16). In addition, any RAD51 k-mer can fall apart in every possible combination of m-mers and n-mers (k = n + m; e.g. a pentamer can fall apart to form a monomer and a tetramer, or a dimer and a trimer). This model provides the concentrations of RAD51 polymers in solution and is solved in the steady-state so that it only depends on a single fit parameter KD (i.e. the RAD51 protomer–protomer dissociation constant). The steady-state assumption is reasonable because in SPR there is a constant flow replenishing any RAD51 that binds to the surface. The two kinetic modules of RAD51 polymer formation on ssDNA and dsDNA were identified by increasing complexity based on mass action kinetics until we identified models that were able to fit the experimental datasets (see Appendix Methods, Appendix Fig S1 and Appendix Table S2 for details). Both the ssDNA and dsDNA modules allow for the adsorption of any RAD51 n-mer from solution onto DNA (provided at least n DNA-binding sites are available), given that n-mers (1 ≤ n ≤ 16) can exist in solution. Polymer elongation occurs when a RAD51 m-mer in solution binds to a DNA-bound n-mer to form a DNA-bound (n + m)-mer, provided at least n + m DNA-binding sites are available and 1 < m + n ≤ 16. However, unbinding was assumed to take place only via single protomer dissociation and via the dissociation of short RAD51 nuclei. This is a valid assumption considering that, in all the SPR experiments in this study, ATP hydrolysis was inhibited due to the presence of Ca2+. This condition would lead to slow RAD51 filament disassembly which can be approximated as disassembly via monomer removal or via the removal of short, unstable RAD51 nuclei. The ssDNA module consists of a polymerisation forward rate constant (kp), an unstable reverse rate constant (ku), a quasi-stable reverse rate constant (kq) and a stable reverse rate constant (ks). kp describes the adsorption and elongation of RAD51 polymers on ssDNA up to a maximum length of 16, ku describes the dissociation of unstable nuclei (one to three RAD51 molecules), kq describes the dissociation of quasi-stable nuclei (four RAD51 molecules), and ks describes the dissociation of single RAD51 protomers (i.e. RAD51 monomers of a RAD51 polymer; Figs 2A and 3A). Similarly, the kinetic module for dsDNA includes kp, ku and ks, but without the need for kq to describe dissociation of quasi-stable nuclei (i.e. two RAD51 molecules; Fig 2C). For dsDNA, ku describes the rate of dissociation of RAD51 monomers not bound to a RAD51 polymer (Fig 3B). Figure 3. Mathematical models describing WT RAD51 polymerisation kinetics on ssDNA and dsDNA A, B. (A) Kinetic representation of the ordinary differential equation (ODE) model describing WT RAD51 polymerisation on ssDNA, consisting of five parameters: kp (polymerisation forward rate), ku (unstable reverse rate), kq (quasi-stable reverse rate), ks (stable reverse rate) and KD (protomer–protomer interaction affinity). KD predicts the concentrations of RAD51 polymers of variable length in solution, while kp, ku, kq and ks predict the speed of formation of RAD51 polymers on ssDNA. (B) Kinetic representation of the ODE model describing WT RAD51 polymerisation on dsDNA, consisting of four parameters: kp, ku, ks and KD. KD predicts the concentrations of RAD51 polymers of variable length in solution, and kp, ku and ks predict the speed of formation of RAD51 polymers on dsDNA. In panels (A, B), purple arrows depict a simplified cartoon representation of RAD51 polymerisation in solution. The model allows for any RAD51 n-mer (1 ≤ n < 16) to associate with any other RAD51 m-mer (1 ≤ m < 16) to form an (m + n)-mer (1 < m + n ≤ 16), and any RAD51 k-mer to dissociate into any combination of n-mers and m-mers (k = n + m). The ssDNA and dsDNA models were calibrated by simultaneously fitting the SPR curves of ssDNA dN-X (dN-8, dN-14, dN-17, dN-50) and dsDNA dN-Xp (dN-5p, dN-8p, dN-11p, dN-50p) using the mode ABC-SMC particles. Mean values ± 1 SD of three mode ABC-SMC particles derived from model fits of three dN-X and dN-Xp repeats (n = 3). ssDNA kp, ku, kq and ks were fit to the ssDNA SPR curves, and dsDNA kp, ku and ks were fit to the dsDNA SPR curves. A single KD was fit to all curves. C, D. (C) Predicted concentrations of WT RAD51 polymers in solution at equilibrium as a function of WT RAD51 monomer concentration (150 nM, 3 μM, 30 μM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. (D) Predicted % WT RAD51 within each polymer state in solution at equilibrium as a function of WT RAD51 monomer concentration (150 nM, 3 μM, 30 μM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. In panels (C, D), the KD was fixed to the mean value identified in Fig 3A and B (i.e. KD = 1.14 nM), and the concentration of WT RAD51 monomer concentration was varied accordingly. In panel (D), the % WT RAD51 values were calculated by multiplying the polymer concentrations by their respective polymer length and dividing each value by the total RAD51 monomer concentration (i.e. % WT RAD51 = [n-mer] * n/[WT RAD51monomer]). [WT RAD51monomer] was calculated via Coomassie staining image quantification using a BSA standard curve. It is important to note that the assumed maximum polymer length in solution (16-mer) leads to an overestimation of the concentration of each n-mer in solution, given that RAD51 is likely to form polymers of length greater than 16 at 150 nM, 3 μM and 30 μM WT RAD51 monomer concentration. However, this overestimation is likely to not alter the conclusion that WT RAD51 forms long polymers in solution at 150 nM–30 μM concentration. Source data are available online for this figure. Download figure Download PowerPoint Using ABC-SMC to fit the aforementioned modules of RAD51 polymers in solution and on DNA to the experimental data of ssDNA dN-X (Fig 2B) and dsDNA dN-Xp (Fig 2D), values for the model parameters were determined (Figs 3A and B, and EV1). This analysis identified a nano-molar range RAD51 protomer–protomer dissociation constant (dN-X and dN-Xp KD = 1.14 ± 0.5 nM) as a key factor driving rapid RAD51 polymerisation on both ssDNA and dsDNA, similarly to other studies using either pressure perturbation fluorescence spectroscopy (Schay et al, 2016) or single molecule fluorescence microscopy (Candelli et al, 2014). This is due to the fact that this KD value enables WT RAD51 to form abundant long polymers in solution at both 150 nM and 3 μM [WT RAD51] (Fig 3C and D). These RAD51 polymers can then directly adsorb onto DNA and elongate, therefore enabling WT RAD51 to polymerise faster on the dN-50 and dN-50p (150 nM [WT RAD51]) compared to the dN-14, dN-17, dN-11p and dN-8p oligonucleotides (3 μM [WT RAD51]) despite a 20-fold lower RAD51 injection concentration. Click here to expand this figure. Figure EV1. ABC-SMC for WT RAD51 SPR data fittingABC-SMC probability densities for the simultaneous WT RAD51 ssDNA (Fig 3A) and dsDNA model (Fig 3B) fits of the ssDNA dN-X (Fig 2B) and dsDNA dN-Xp (Fig 2D) SPR data. Heat maps indicate particle frequencies and describe any pairwise correlations between model parameters (dN-X & dN-Xp KD, dN-X kp, ku, kq, ks, dN-Xp kp, ku, ks). All heat map axes are in log–log scale and are labelled according to the corresponding parameter colour: dN-X & dN-Xp KD (purple), dN-X kp (orange), dN-X ku (brown), dN-X kq (black), dN-X ks (green), dN-Xp kp (yellow), dN-Xp ku (cyan) and dN-Xp ks (blue). A high particle frequency corresponds to a good model fit to the data for the specified parameter pair. The probability densities for individual parameters are presented along the top left diagonal and were estimated using a Gaussian kernel. Particles were initialised via log-uniform priors with the following lower and upper bounds: 10−4 < dN-X & dN-Xp KD < 10−1, 10−4 < dN-X kp < 104, 10−6 < dN-X ku < 103, 10−6 < dN-X kq < 103, 10−6 < dN-X ks < 103, 10−4 < dN-Xp kp < 104, 10−6 < dN-Xp ku < 103 and 10−6 < dN-Xp ks < 103. PD, probability density. Source data are available online for this figure. Download figure Download PowerPoint Importantly, we also identified a 6.5-fold higher ssDNA polymerisation forward rate constant (dN-X kp = (2.6 ± 0.18) × 10−2/μM/s) compared to dsDNA (dN-Xp kp = (4 ± 0.6) × 10−3/μM/s), explaining the overall faster RAD51 polymerisation on ssDNA (Fig 3A and B). Together, these analyses suggest that WT RAD51 adsorption and/or elongation is faster on ssDNA compared to dsDNA, and that a high protomer–protomer affinity enables WT RAD51 to nucleate and elongate effectively on ssDNA and dsDNA even at low concentrations of RAD51. RAD51 polymerises faster on flexible DNA Overall, our analyses suggest that WT RAD51 can nucleate more efficiently on short dsDNA molecules but polymerises significantly faster on long ssDNA molecules. We speculate that the difference in RAD51 polymerisation speed is due to the higher flexibility of ssDNA compared to dsDNA. Explicitly, we propose a Bend-To-Capture (BTC) mechanism to explain how DNA flexibility can impact polymerisation kinetics (Fig 4A). A free RAD51 monomer or polymer in solution would need to generate two sequential, non-covalent interactions to be incorporated into the growing polymer: the interaction with the exposed interface of an existing RAD51 polymer and with the exposed scaffold DNA. In order for the incoming RAD51 to fit into the position without steric clashes, we reasoned that naked DNA immediately next to the existing RAD51 polymer needs to be in a configuration that can bend away from the preferred direction of the polymer. In this way, flexible DNA is expected to explore more conformations compatible with the further addition of RAD51 per unit time. Figure 4. Kinetics of WT RAD51 binding to DNA of varying flexibility A. A depiction of the Bend-to-Capture (BTC) mechanism. RAD51 generates two sequential, non-covalent interactions (a RAD51 protomer–protomer interaction and a RAD51-DNA interaction, or vice versa) faster on flexible DNA (depicted as ssDNA) compared to rigid DNA (depicted as dsDNA). The free RAD51 monomer or polymer in solution (here depicted as a monomer) to be incorporated into the growing polymer is shown with an asterisk. ΔTS is the entropic penalty of restricting DNA bending fluctuations, which depends on DNA persistence length. ΔGRAD51-RAD51 and ΔGRAD51-DNA are binding energies, which do not depend on DNA persistence length, i.e. degrees of freedom within the macrostate of being bound or unbound. B. Biotinylated DNA molecules of varying flexibility were separately immobilised onto SPR CM5 chips via biotin–streptavidin interaction, and WT RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics. The flexibility of respective DNA, the expected entropic penalties upon RAD51 binding to corresponding DNA and protomer–protomer binding energy contribution of WT RAD51 are indicated in grey, green and blue boxes. H and L in each box denote high and low, respectively. C. The mean number of WT RAD51 bound to respective DNA oligonucleotides, as measured by SPR (dotted lines) and ODE model fits (solid lines). D, E. Bar plots of fitted kp values (D) and ks values (E) for each SPR curves. In panel (D), all parameters except kp were fixed to the mean values as identified in Fig 3A and B, and kp was fitted using lsqcurvefit (MATLAB). In panel (E), all parameters except ks and kp were fixed to the mean values as identified in Fig 3A and B, and ks and kp were fitted using lsqcurvefit (MATLAB). Only the ks values are reported here. Mean ± 1 SD of three ODE model fits (n = 3). (D): un-paired, one-tailed Mann–Whitney–Wilcoxon tests. (E): un-paired, two-tailed Mann–Whitney–Wilcoxon tests. *P ≤ 0.05; ns, non-significant. Source data are available online for this figure. Download figure Download PowerPoint To test this notion, we designed an experiment to measure the kinetics of WT RAD51 binding to DNA oligonucleotides of varying flexibility. It has been shown that poly-dT ssDNA, which is widely used for RAD51 binding assays, is highly flexible, while poly-dA ssDNA is highly rigid due to base stacking interactions (Mills et al, 1999; Sim et al, 2012). Consistently, our SAXS-derived persistence length (Lp) measurements showed ssDNA dT-50 is the most flexible oligonucleotide, followed by dN-50, dA-50 and dsDNA dN-50p (Fig EV2 and Table 2). By measuring RAD51 binding kinetics to these DNA oligonucleotides (Fig 4B and Table 1), we found that WT RAD51 indeed displayed faster association with the dT-50 compared to the dN-50, and the model fit suggests this is due to a higher polymerisation rate constant (kp; Fig 4C and D). Furthermore, WT RAD51 displayed very slow association with dA-50, comparable to that o
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