PMC:4103454 / 10337-14559 JSONTXT

{"project":"2_test","denotations":[{"id":"24981866-11389467-20021739","span":{"begin":309,"end":313},"obj":"11389467"},{"id":"24981866-9223630-20021740","span":{"begin":1113,"end":1117},"obj":"9223630"},{"id":"24981866-11240135-20021741","span":{"begin":1924,"end":1928},"obj":"11240135"}],"text":"VRR-Nuc Domains Are Dimeric and Cleave Four-Way DNA Junctions\nA key characteristic of canonical HJRs is that they bind and process their substrate as dimers to achieve substrate recognition and productive resolution and predominantly form homodimers in solution in the absence of substrate (Lilley and White, 2001). We examined the quaternary structure of the VRR-Nuc domains in solution by performing analytical ultracentrifugation (AUC) experiments (Figure 3A). For each protein, the main peak in the sedimentation coefficient distribution analysis (C(S)) corresponded to a dimer. For psNUC, an additional minor peak corresponding to a tetrameric species was present at concentrations above 70 μM. This agrees well with our crystal structures and the known quaternary structure of HJRs.\nThe ability of VRR-Nuc domains to bind a range of DNA structures was examined using electrophoretic mobility-shift assays. As shown in Figure 3B, psNUC bound a four-way DNA junction stoichiometrically with an affinity indicative of a subnanomolar equilibrium dissociation constant (KD) typical of HJRs (∼1 nM; White et al., 1997) whereas stNUC bound the junction over 100 times more weakly (KD = 200 nM). Nevertheless, in both cases, the binding was structure-selective, with psNUC forming weaker complexes with a 5′ flap and a nicked three-way junction and stNUC binding the four-way junction only (Figure S3A). At high protein concentrations, higher-order complexes formed with equal efficiency on all substrates. By contrast to the other proteins, saNUC only formed nonspecific, higher-order complexes.\nWe next investigated the catalytic activity of the VRR-Nuc domains toward these DNA substrates. Figure 3C shows the cleavage products obtained with psNUC and Jbm5, a four-way junction that can undergo 12 steps of branch migration such that each possible dinucleotide can be accommodated at the branchpoint (Kvaratskhelia et al., 2001). This junction was cleaved symmetrically, and we observed only two discrete cleavage sites on each strand that occurred within or close to the homologous core of the junction (boxed, Figure 3D). This resulted in the formation of ligatable nicked duplex DNA, diagnostic of productive HJ resolution (Figure S3B). Under the same assay conditions, we observed no cleavage of linear double-stranded DNA substrate and very weak activity on most branched DNA structures with the exception of replication forks, which were cleaved to an intermediate degree (Figure S3C). Finally, psNUC cleaved a fixed HJ 3 nt 3′ to the branchpoint, and this activity was inhibited by the presence of a slow-cutting mutant of T7 endonuclease I, suggesting that they compete for the branchpoint (Figure S3D).\nstNUC and saNUC exhibited weaker nuclease activity against the Jbm5 HJ substrate, detectable only at protein concentrations above 1 μM and with increased reaction times (Figure 3E). Under these conditions, stNUC showed robust activity, whereas cleavage by saNUC was much reduced. This suggests that our saNUC preparations have a low active fraction potentially also explaining the lack of structure selectivity seen in the gel mobility shift experiments. In both cases, we observed a range of symmetrical cleavage sites within or close to the homologous core of the junction as well as some nonsymmetrical sites away from the branchpoint (Figure 3F). stNUC also selectively cleaved a nicked three-way junction (RF) and a 3′ flap structure (3′F) 2 bp 3′ to the branchpoint (Figure S3E). Both enzymes additionally exhibited a nonspecific nuclease activity producing a ladder of cleavage products (Figure S3F).\npsNUC therefore not only structurally resembles an archaeal HJR but also behaves as one, displaying binding to and symmetrical cutting of HJ substrates with affinities and cleavage patterns typical of a canonical HJC protein. Whether the levels of activity observed for stNUC and saNUC are representative of those in vivo is unclear, but it would appear unlikely given their close structural homology to psNUC. Crucially, however, both enzymes retain the ability to produce symmetric cleavages within a HJ substrate, an activity that is not characteristic of VRR-Nuc domains within the FAN1 orthologs."}