Organization of the BcgI restriction-modification protein for the cleavage of eight phosphodiester bonds in DNA

Abstract

Type IIB restriction-modification systems, such as BcgI, feature a single protein with both endonuclease and methyltransferase activities. Type IIB nucleases require two recognition sites and cut both strands on both sides of their unmodified sites. BcgI cuts all eight target phosphodiester bonds before dissociation. The BcgI protein contains A and B polypeptides in a 2:1 ratio: A has one catalytic centre for each activity; B recognizes the DNA. We show here that BcgI is organized as A(2)B protomers, with B at its centre, but that these protomers self-associate to assemblies containing several A(2)B units. Moreover, like the well known FokI nuclease, BcgI bound to its site has to recruit additional protomers before it can cut DNA. DNA-bound BcgI can alternatively be activated by excess A subunits, much like the activation of FokI by its catalytic domain. Eight A subunits, each with one centre for nuclease activity, are presumably needed to cut the eight bonds cleaved by BcgI. Its nuclease reaction may thus involve two A(2)B units, each bound to a recognition site, with two more A(2)B units bridging the complexes by protein-protein interactions between the nuclease domains.

Figure 1.

Native MS. A nano-ESI mass spectrum of the E53A form of BcgI (27.5 µM) in 200 mM AmAc was recorded under non-denaturing conditions (see ‘Materials and Methods’ section). The insert shows the signals for the oligomeric states in the m/z range from 8500 to 14 500 magnified 5-fold. The charge state series assigned to each molecular species (in bold) are indicated with dashed brackets, and the charge and m/z value for the primary peak in each series is given.

Figure 2.

Sedimentation equilibrium. Samples of BcgI in AUC buffer were centrifuged at 7500 rpm at 20°C for the times noted below. The main panels show the absorbance (white circles) as a function of the centrifugal radius: (a) 0.44 µM BcgI at 230 nm after 30 h; (b) 18 µM BcgI at 292 nm after 44 h. The lines through the data points in the main panels are the best fits of the following data sets to Equation 1: (a) the records after 26 and 30 h; (b) after 32, 38 and 44 h. The best fits were obtained with values for M of 183 and 431 kDa in (a) and (b) respectively. The plots above the main panels display the residuals between these fits and the experimental data: the random variations in (a) show that the record can be accounted for by a single ideal species with a unique value of M; the systematic deviations in (b) demonstrate that more than one species is present, which gives rise to a value.

Figure 3.

Self-association of BcgI. Varied concentrations of BcgI in AUC buffer were loaded into the AUC and sedimented to equilibrium at 7500 rpm at 20°C. For each sample, the absorbance was measured as a function of centrifugal radius and fitted to Equation 1. Apart from the data at the lowest BcgI concentrations (viz. Figure 2a), the residuals between the fits and the experimental data displayed non-random deviations (viz. Figure 2b), so the best fit at each initial concentration denotes an value. The values are plotted against the loading concentrations of BcgI. The values expected for the 182.4 kDa A₂B protomer and for the 365 kDa (A₂B)₂ dimer are marked with long and short dashes, respectively.

Figure 4.

AUC of BcgI–DNA complexes. The samples, in AUC buffer at 20°C, contained WT BcgI protein (7.5 µM) and a DNA duplex (2 µM) as follows: (a) HEX-42NS, a non-specific DNA that lacks the recognition sequence; (b) HEX-42S, a specific DNA with both recognition and cleavage loci. The samples were centrifuged in the AUC and the absorbance across the cell monitored at 539 nm at the following stages after initiating the run: the instrument was initially set to spin at 3000 rpm and an absorbance scan recorded immediately on reaching that speed (black trace). The velocity was then raised to 7500 rpm and scans taken after 14 h and after further 8 h delays. The records shown are after 14 h (green trace), 22 h (cyan trace), 38 h (pink trace) and 54 h (blue trace). The data sets with the non-specific duplex (a) recorded after ≥30 h were fitted to Equation 1: the best fit gave a of 349 kDa.

Figure 5.

DNA cleavage at low [BcgI]. Reactions in buffer R at 37°C contained one of the following plasmids (³H-labelled, initially ∼90% SC monomer) at 5 nM and BcgI protein at the concentrations noted below. The plasmids were (a) pUC19, which has one BcgI site; (b) pDG5, which has two sites. The enzyme concentrations for each reaction are marked on the right: 2 nM, white circles; 5 nM, black circles; 10 nM, white squares; 20 nM, black squares. Samples were taken at the indicated times and analysed by electrophoresis to separate the SC substrate from the cleaved products. The residual concentrations of the SC DNA were determined and are plotted as a function of time. The lines are the best fits to single exponential decays with floating offsets.

Figure 6.

DNA cleavage at high [BcgI]. (a) The reactions, in buffer R at 37°C, contained 5 nM ³H-labelled SC pUC19 and BcgI protein at 75 nM, white circles; 250 nM, black circles; 750 nM, white squares. After manually mixing the reagents, samples were taken from the reactions by pipette and mixed immediately with an EDTA quench. The samples were then subjected to electrophoresis through agarose, to enable the concentration of SC DNA to be determined. The amount of SC DNA present in each sample is plotted against the time. (b) As (a) except that the DNA was SC pDG5 and that the reactions were carried out by quench-flow. (c) Apparent rate constants (k_obs) from ≥3 independent experiments at each BcgI concentration were averaged and plotted against the concentration: for reactions on pUC19, white circles; for reactions on pDG5, black circles. Error bars denote standard deviations. The line drawn through each data set is the best fit to a hyperbolic function, k_obs = (k_max × [BcgI])/(K_½ + [BcgI]). The best fits were obtained with the following: for pUC19, k_max = 2.7 min⁻¹ and K_½ = 232 nM; for pDG5, k_max = 21 min⁻¹ and K_½ = 230 nM.

Figure 7.

DNA binding. (a and b) The mixtures contained, in 10 µl buffer R*C, 20 nM DNA, either HEX-42S (a) or HEX-42NS (b), and BcgI at one of the following levels, from left to right: 0, 20, 40, 60, 80, 100, 150 or 200 nM. After 5 min at 37°C, the samples were subjected to electrophoresis through polyacrylamide and the fluorescence from each band recorded. On the left of gel images, F marks the position of the free DNA; C, the DNA–protein complexes that entered the gel; and W, the bottom of the wells. (c) From titrations of both HEX-42S and HEX-42NS with increasing amounts of BcgI protein, and from additional experiments with 24 bp duplexes (HEX-24S and HEX-24NS: gels not shown), the amount of free DNA in each sample was determined relative to the amount in a parallel lane without enzyme. The % DNA in the free form is shown as a function of the BcgI concentration: HEX-42S, white triangle; HEX-42NS, white squares; HEX-24S, black triangles; HEX-24NS, black squares. Mean values from three repeats are given: error bars show standard deviations. The red lines through the data with the specific duplexes denote stoichiometric binding where all of the added BcgI is bound to DNA until saturation of the DNA is achieved.

Figure 8.

Activation of BcgI by BcgIA. The reactions, in 200 µl buffer R* at 37°C, contained 100 nM BcgIA protein and 5 nM ³H-labelled pUC19 (initially 90% SC monomer), in either the absence or presence of 5 nM WT BcgI. Aliquots (20 µl) were removed at the times shown and immediately quenched before analysis by electrophoresis: one-half through agarose, to separate the SC, OC and LIN forms of the plasmid; the other half through polyacrylamide, to capture the 34-mer. The concentrations of the various forms of pUC19 were evaluated from the agarose gel and that for the 34-mer from the polyacrylamide gel (see ‘Materials and Methods’ section). For the reaction with BcgIA alone, only the level of SC DNA is shown: red unfilled triangles and line. For the reaction with both BcgIA and native BcgI, the following were assessed: intact SC DNA, white circles; nicked OC DNA, white squares; LIN DNA, with at least one DSB at the BcgI site, white inverted triangles; the 34-mer, black triangles. Mean values from three repeats are shown: error bars denote standard deviations.

Figure 9.

Scheme for action at eight phosphodiester bonds by BcgI. (a) The substrate for the REase activity of BcgI consists of two copies of its recognition sequence. The sites, shown here aligned in parallel, can be located either on the same DNA molecule, in cis, or on separate molecules, in trans. The bipartite recognition site is indicated by black segments, with an arrowhead to mark its 5′–3′ orientation: strand polarities are also indicated at DNA termini. Cleavage loci 10 and 12 nt distant from the site are marked with vertical arrows: upstream and downstream loci are noted with − or + signs, respectively. (b) The two sites in (a) are both shown bound to one A₂B protomer of BcgI: an interaction between the protomers is suggested by the double-headed arrow (in red). The B subunit (yellow oval) is responsible for DNA specificity and is positioned spanning both segments of the recognition sequence. The A subunits carry both REase and MTase activities and are illustrated as separate domains connected by a flexible linker. The MTase domains (green squares) overlay the sites of methylation in the specified segments of the recognition sequence. The REase domains (cyan triangles) are placed against the target bonds at the −10 and +10 positions, upstream and downstream of the site in top and bottom strands, respectively: to mark their orientation on the DNA, the triangle points towards the 5′-end of each strand. At this stage, BcgI has yet to engage the scissile bonds at the −12 and +12 positions. (c) As (b) except that two additional A₂B units from free solution now bridge the A₂B units bound to each site via interactions between the nuclease domains of the free and the DNA-bound units. The two nuclease domains from one of the extra A₂B units engage the scissile bonds at the −12 positions upstream of both recognition sites (on the left, as drawn in [c]) while those from the other occupy the downstream +12 positions in both sites (on the right). The scheme thus results in a dimer of nuclease domains, with two catalytic centres for phosphodiester hydrolysis at all four loci where BcgI makes a DSB.