RAD51 unwinds dsDNA [4, 17], but the underlying mechanism of this process with respect to biochemical reaction conditions is far from clear. We have addressed this aspect in our current study. Here we delineate specific nucleotide cofactor requirements for DNA unwinding and provide mechanistic insights on how RAD52 collaborates with RAD51 in this process. Furthermore, we show that unwinding of duplex DNA by RAD51 correlates positively with aggregation of RAD51-dsDNA complexes.
RAD51 mediated dsDNA unwinding does not require ATP hydrolysis
Fully relaxed circular dsDNA was generated from φX174 supercoiled plasmid by the action of Drosophila Topo I. Subsequent unwinding of relaxed dsDNA by RAD51 was assayed in the presence of Topo I, followed by deproteinization and agarose gel electrophoresis (Methods). DNA unwinding as a function of RAD51 concentration depended entirely on nucleotide cofactor conditions. ATP as well as its poorly/non-hydrolysable analogues such as ATPγS/AMP-PNP facilitated efficient unwinding (lanes 1–3, Panels C, F & E, Fig. 1), while the absence of nucleotide cofactor failed in the same (lanes 1–3, Panel H, Fig. 1). Surprisingly, detectable level of unwinding was observed even in presence of ADP (lanes 1–3, Panel D, Fig. 1), whereas with AMP no unwinding was observed (lanes 1–3, Panel G, Fig. 1). Further quantification of the gel revealed, maximal DNA unwinding (~44%) even at the lowest RAD51 concentration [3 μM] in the presence of ATP/AMP-PNP, whereas DNA unwinding increased as a function of RAD51 concentration in ATPγS/ADP conditions and reached about 34% and 22% respectively at maximal protein concentration (9 μM) (Fig. 1I). Thus unwinding appears to be ATP binding but not hydrolysis dependent, as not only ATP but also its non-hydrolysable analogs support the process, which is in congruence with previously published reports [4] and [17]. However, even product of ATP hydrolysis, ADP was seen to support DNA unwinding by RAD51, a result that appears somewhat counter-intuitive at the outset. But in fact, even this observation is consistent with earlier result that RAD51 binds ssDNA sufficiently well that it promotes strand annealing even in ADP conditions, a property starkly contrasting with that of well studied E. coli RecA protein [35]. Furthermore, preliminary linear dichroism experiments with RAD51, calf-thymus dsDNA and ADP have confirmed that the HsRad51-dsDNA complex is also stable in the presence of ADP (H.K. Kim, K. Morimatsu & B. Nordén, unpublished observations). Thus, ADP stabilizes both ssDNA and dsDNA complex formation with HsRad51, although it does not promote the strand exchange reaction. This could suggest that the role of the nucleotide cofactors is not only to stabilize or destabilize the protein-DNA complex, but also involves changing the conformation of the complex to promote the strand exchange reaction. In order to eliminate the possibility that unwinding observed in ADP condition is not a trivial consequence of either ATP contamination or artifactual conversion of ADP to ATP, we assessed the integrity of ADP by Thin Layer Chromatography (TLC) assay [36]. Expectedly we found that ADP containing samples showed no traces of ATP contamination. Added ADP was pure and no artifactual conversion to ATP had happened (data not shown). Therefore we strongly believe that ADP effects were genuine.
RAD52 stimulates RAD51 mediated duplex unwinding
In order to test the role of RAD52 in DNA unwinding, we titrated the reactions with increasing levels of RAD52 while maintaining RAD51 concentration constant at a lower level. As expected, conditions that failed to promote RAD51 mediated unwinding, namely absence of nucleotide cofactor or presence of AMP, remained ineffective even with addition of RAD52 (lanes 2–4, Panels F & E, Fig. 2). On the other hand, nucleotide cofactor conditions that are congenial for RAD51 mediated unwinding, namely ATP/AMP-PNP/ATPγS/ADP, revealed marginal and detectable improvement in unwinding following RAD52 addition (lanes 2–4, Panels A, C, D & B, Fig. 2), while RAD52 alone did not lead to any detectable unwinding in the presence or absence of any of these nucleotide cofactors (lane 5, Panels A-F & lanes 1–3, Panel G, Fig. 2). Moreover, such RAD52 mediated enhancement of DNA unwinding by RAD51 was evident even in presence of ADP, when time-course of unwinding was studied. At fixed concentrations of RAD51 and RAD52, unwinding efficiency with ADP, reached a level similar to that of ATP, within 10 minutes of incubation (lane 4, Panel B, Fig. 3) (Fig. 3C). On the contrary, ADP containing reaction that lacked RAD52, showed only marginal unwinding which hardly increased with time (only last time-point is shown; lane 7, Panel B, Fig. 3) (Fig. 3C).
RAD52 forms large RAD52-RAD51-DNA complexes
To understand molecular basis of RAD52 mediated stimulation on DNA unwinding by RAD51, we carried out DNA binding studies of RAD51 and RAD52 with circular dsDNA in the presence of various nucleotide cofactors. RAD51 protein when bound to dsDNA, generated distinctly slower migrating gel-shifted complexes as a function of increasing protein concentration. This was so in all nucleotide conditions, suggesting RAD51 binding to dsDNA was not dependent on nucleotide cofactor conditions (lanes 2–4 & 8–10, Panel A: lanes 1–3 & 7–9, Panels B & C, Fig. 4) which is consistent with previously published reports [17]. Under same conditions, binding of RAD52 alone to dsDNA generated large protein-DNA complexes that failed to enter the gel (Panel D, Fig. 4) as reported earlier [37]. Interestingly, we find that, addition of RAD52 converted RAD51-DNA gel-shifted complexes also into large complexes that fail to enter the gel (lanes 5–7 & 11–13, Panel A: lanes 4–6 and 10–12, Panels B & C, Fig. 4). Irrespective of the nucleotide cofactor conditions, addition of RAD52 resulted in large RAD52-RAD51-DNA complexes. We have further confirmed that these large protein-DNA complexes entrapped in the wells indeed contain both RAD51 and RAD52 proteins by excising agarose gel fragments containing these complexes, and subjecting them to protein compositional analyses by SDS-PAGE (Data shown only for complex formed in ATP conditions, the boxed region of lane 5, analyzed in lane 1, Panel E, Fig. 4).
RAD52 co-aggregates with RAD51 on DNA
In order to corroborate that these complexes indeed house both proteins and DNA, we carried out centrifugation assay and tested the ability of RAD52 to "co-aggregate" both RAD51 and itself onto DNA. Also, the same was analyzed with respect to ongoing DNA unwinding reaction. RAD51 protein, under the current experimental conditions, predominantly stayed in supernatant while a minor fraction sedimented following centrifugation assay (lane s1, Panel D, Fig. 5). On the contrary, RAD52 protein that is largely in aggregated state by itself (lane p, Panel B, Fig. 5), increases aggregation prone ness of RAD51 protein in its presence (lanes p1 and s1, Panel C, Fig. 5) where RAD52 protein retains its high state of aggregation (lane p1, Panel C, Fig. 5). Furthermore, we find RAD51-dsDNA complexes that are largely soluble in ATP/ADP conditions (lanes s1, Panels E & F, Fig. 5) get aggregated with addition of RAD52. This is evidenced by increase in levels of RAD51 protein (lanes p2–p4, Panels E & F, Fig. 5) as well as relaxed/unwound dsDNA (lanes p2–p4 Panels D & E, Fig. 6) in pellet fraction as a function of RAD52. Even in other conditions, namely, AMP-PNP/ATPγS/AMP or in the absence of any nucleotide cofactor, a similar trend is observed, where RAD52 protein renders RAD51-dsDNA complexes, aggregation prone (Figs. 5 &6). Protein (lanes p2–p4, Panels G-J, Fig. 5) as well as DNA (lanes p2–p4, Panels F-H, Fig. 6) analyses reveal the trend that complexes comprising of RAD51/RAD52/dsDNA were "aggregated" as a function of RAD52 concentration. We speculate that these complexes in fact, may represent "co-aggregates" as both proteins are known to interact with each other in these reaction conditions [38]. Interestingly, DNA analyses reveal that "co-aggregates" contained not only substrate DNA i.e. relaxed dsDNA, but also products of unwinding. Quantitative analysis of proteins as well as DNA in the gel assays revealed that in all conditions of nucleotide cofactors tested, RAD51 protein and dsDNA co-sedimented as a function of RAD52 concentration. This is evidenced by an increase in recovery of RAD51 protein as well as dsDNA in pellet fractions with a concomitant depletion of the same in supernatant fractions [Fig. 5K: Protein analysis (data shown only for pellet fractions) & (Fig. 6I: DNA analysis (only pellet fraction data is shown)].
Salt (KCl) induced changes in aggregation of RAD51-DNA complexes
In order to unravel any mechanistic link between RAD51-dsDNA aggregation and duplex unwinding, we probed the system by salt titration study. Moreover, we also tested whether salt induced aggregation of RAD51-dsDNA complexes would then obviate the need of RAD52 in the same. Centrifugation analysis of RAD51-DNA complexes as a function of varying KCl concentration in presence of ATP revealed interesting trends: RAD51-dsDNA aggregation increased in low salt (up to 100 mM KCl), followed by a drop at high salt regime (100–200 mM KCl) [(Fig. 7A: protein analysis & Fig. 7C: DNA analysis) (data shown only for pellet fractions)]. This trend was unaffected by the presence of RAD52 [(Fig. 7B: protein analysis) & (Fig. 7D: DNA analysis)]. Quantification of gel data revealed increase in RAD51 and dsDNA levels in pellet fractions as a function of salt concentration (up to 100 mM), followed by a decline of the same at higher salt concentration (100–200 mM) (Fig. 7E). Concomitantly, supernatant fractions revealed reciprocal trend. On the contrary, under same reaction conditions, RAD52 protein showed a different trend: RAD52 protein that was highly aggregated to begin with (without salt), disaggregated with the addition of salt. Salt induced disaggregation of RAD52 ensued from > 50 mM KCl onwards, and reached completion at about 140 mM KCl (Figs. 7B &7E).
Salt (KCl) induced changes in dsDNA unwinding by RAD51
In an attempt to correlate salt (KCl) induced changes in aggregation of RAD51-dsDNA complexes with corresponding changes in DNA unwinding, we analyzed RAD51 mediated unwinding at varying concentrations of KCl. Quantitative analysis of gel assay revealed that unwinding was stimulated in low salt concentration (reaching up to 100–120 mM KCl), followed by a fall in the same at higher KCl concentration (> 100 mM KCl) (Fig. 8). In fact, by about 160 mM KCl, the unwinding level was barely detectable. By and large the effect of salt (KCl) on RAD51 mediated unwinding was very similar even in presence of RAD52. Consequently, the salt titration studies suggested that low concentration of salt (up to 100 mM) which is conducive for RAD51-dsDNA aggregation, in parallel, also resulted in an increase in dsDNA unwinding. Conversely KCl at high concentration (> 100 mM) led to decrease in aggregation as well as dsDNA unwinding. RAD52 protein in RAD52-RAD51-dsDNA complexes started disaggregating at concentrations of KCl greater than 50 mM (Figs. 7B &7E), thereby suggesting that changes in RAD51-dsDNA aggregation as well as dsDNA unwinding are relatively independent of RAD52 protein in the same regime of salt concentration. Accordingly, these experiments further suggest that process of dsDNA unwinding is mechanistically linked to the aggregation state of RAD51 in RAD51-dsDNA complexes.
In order to test whether enhanced aggregation as well as dsDNA unwinding observed in parallel at low salt (up to 100 mM KCl) was also accompanied by any changes in RAD51 binding to dsDNA, we analyzed the samples by gel-shift assays. Since the observed effects of salt were independent of RAD52 (as described above), we analyzed RAD51-dsDNA complexes as a function of KCl in the absence of RAD52. Gel-shift analyses revealed that mobility of protein-DNA complexes increased as a function of salt, thereby suggesting that RAD51 binding to dsDNA was reduced in the presence of salt. At high concentration of salt [200 mM] the protein-DNA complexes appeared to have dissociated, giving rise to the release of free dsDNA (lane 7, Fig. 9).