Characterisation of theType IIB restriction enzyme BcgI

1. Characterisation of theType IIB restriction enzyme BcgI Sumita Ganguly, Jacqueline T. Marshall, Darren M. Gowers and Stephen E. Halford The DNA-protein Interactions Unit, Department of Biochemistry, School of Medical Sciences University of Bristol, Bristol BS8 1TD, United Kingdom

2. nnnnnnnnnnnnnnCGAnnnnnnTGCnnnnnnnnnnnnnn nnnnnnnnnnnnnnGCTnnnnnnACGnnnnnnnnnnnnnn The Type IIB enzyme BcgI BcgI is a Type IIB restriction enzyme that recognises a discontinuous asymmetric DNA sequence and in the presence of Mg2+ and S-adenosylmethionine (SAM), it cleaves the sequence bilaterally and symmetrically, to release a 34 bp fragment containing the intact recognition sequence (1) as shown below (Fig. 1). After excising the 34 bp fragment, BcgI continues to methylate the fragment. Figure 1.Recognition and cleavage sites of BcgI. The bases shown in red indicate the recognition site of BcgI whereas the green lines indicate the points of cleavage by BcgI.

3. N N C C 341 637 Restriction domain (requires Mg2+ and some Type IIBs require SAM) Methylation domain (requires SAM) Substrate DNA recognition domain (requires target DNA) (A2B) The BcgI subunits The BcgI Restriction-Modification system consists of two adjacent genes, A and B, in the same orientation (2). Mutation studies have shown that the A subunit is responsible for both restriction endonuclease and modification methyltransferase activities whereas the B subunit is responsible for DNA recognition (Fig. 2). Neither protein can cleave or modify DNA on its own but together they form a complex of composition A2B that can perform both functions. However, it is not known how the enzyme co-ordinates these competing activities (3). BcgIA = Restriction-ModificationBcgIB = DNA recognition Figure 2. BcgI subunits A and B. The N-terminal part of the A subunit is believed to be responsible for the endonuclease activity whereas the C-terminal part constitutes the methylation domain. The BcgIB subunit is believed to be responsible for DNA recognition.

4. One-site plasmid Two-site plasmid Two-site catenane 5 Supercoiled Supercoiled 4 Cut in both rings Cut at 1 site Cut at both sites 3 DNA (nM) 2 Cut at 1 site Cut in one ring 1 oc 0 0 20 40 60 0 20 40 60 0 20 40 60 Time (min) BcgI cleavage on one and two-site DNA Previous experiments have shown that BcgI cleaves DNA with two BcgI sites faster than DNA with one BcgI site and that the enzyme also acts concertedly on the two-site DNA, to release as the initial products the DNA cut at both sites. With catenated substrates containing one BcgI site in each ring, the DNA is cleaved as rapidly as the two-site plasmid, to yield directly the DNA products cut in both rings. Hence, the enzyme must act at two sites through 3D space, as opposed to following the 1D path along the DNA (Fig. 3). Figure 3. BcgI on plasmids containing respectively one or two BcgI sites and on two-site catenanes. The reactions, at 37 ºC, contained 5 nM 3H-labelled DNA and 2 Units of BcgI in BcgI reaction buffer. Following the addition of the enzyme to the DNA, aliquots were removed at timed intervals, analyzed by electrophoresis through agarose, and the concentrations of each form of the DNA determined.

5. A. Low Enzyme (1 µM) B. Intermediate enzyme (2.5 µM) C. High enzyme (5.4 µM) 1 4 10 0 Residuals Residuals Residuals 0 0 -1 -4 -2 -10 0.6 0.8 1.5 0.6 Absorbance at 280 nm 0.4 1.0 Absorbance at 230 nm Absorbance at 230 nm 0.4 0.2 0.5 0.2 0.0 0.0 0.0 5.85 5.90 5.95 6.00 6.05 6.10 6.30 6.35 6.40 6.45 6.50 6.55 6.35 6.40 6.45 6.50 6.55 6.60 6.65 Radius Radius Radius Speed = 8000 rpm, MW = 176,836 Speed = 9500 rpm, MW = 184,974 Speed = 5500 rpm, MW = 605,018 Analytical Ultracentrifugation to determine subunit composition of BcgI AUC has been performed on BcgI at three different enzyme concentrations. At low concentration, it gives a MW consistent with a trimer of composition A2B (Fig. 4A). At intermediate concentrations, the protein started to aggregate, as indicated by the systemic variation in the residual fits (Fig. 4B). At high concentrations, large aggregates were formed (Fig. 4C). At low concentrations, no differences were observed in the presence or absence of SAM and/or Mg2+ (data not shown), thus confirming BcgI as an A2B trimer. Figure 4. Graphs of absorbance against centrifugal radius, obtained for BcgI at three different enzyme concentrations in the AUC at 20 ºC (lower panels). Red lines indicate the best fits to single MW values and the residuals are indicated in the upper plots.

6. A) DNA with one BcgI site 5 B) C) 4 3 DNA (nM) 2 Add cognate oligo BcgI enzyme 1 0 1 10 100 1000 0 Trans reaction Oligoduplex (nM) Oligoduplex (nM) ataCGAccgagtTGCtct HEX-ataCGAccgagtTGCtct Figure 5. A) mechanism of “oligo-activation”. B) and C), activation using unlabelled (B) and Hex-labelled (C) 18-mers, sequences as shown with the BcgI recognition sequence in red. The reactions at 37 ºC in BcgI buffer contained 8 nM BcgI, 5 nM 1-site plasmid and either no oligo or oligo as indicated. After 20 min, the reactions were stopped and the concentrations of the SC substrate ( ), the OC intermediate ( ), and the linear product ( ) were determined. Add more oligo 5 4 3 2 1 0 1 10 100 1000 0 Trans activation of reactions on 1-site DNA In order to find the shortest oligonucleotide containing the BcgI recognition site for further AUC experiments, the cleavage of the one-site plasmid was measured in the presence of varied concentrations of both Hex-labelled and unlabelled oligonucleotides of different lengths. The shortest tested were 18 bp long, containing the recognition site but without the cleavage sites. Enhanced plasmid cleavage was observed with both unlabelled and Hex-labelled 18 mers (Fig. 5).

7. 0.22 0.22 A) B) 0.2 0.2 Specific oligo Kd = 72 nM, ΔAnis = 0.12 0.18 EDTA buffer Kd = 261 nM 0.18 Anisotropy Anisotropy 0.16 0.16 Ca2+ buffer Kd = 72 nM 0.14 0.14 Non-specific oligo Kd = 112 nM, ΔAnis = 0.06 0.12 0.12 0.1 0.1 600 0 200 400 0 200 400 600 [E] (nM) [E] (nM) Anisotropy measurements using Hex-labelled oligonucleotides Fluorescence anisotropy experiments with BcgI using the HEX-labelled specific oligonucleotide, revealed similar anisotropy changes in both Ca2+ and EDTA buffers, but tighter binding in the presence of Ca2+ (Fig. 6A). However, in Ca2+ buffers, the specific and non-specific oligos bound with similar affinities but the specific gave a much larger anisotropy change than the non-specific oligonucleotide (Fig. 6B). Hence, when bound to BcgI, the non-specific oligo must retain more mobility than the specific oligo. Specific –HEX-ataCGAccgagtTGCtct Non-specific - HEX-ataCGTccgagtAGCtct Figure 6. (A) Fluorescence anisotropy of the specific HEX-labelled 18-mer (20 nM) in the presence of increasing concentrations of BcgI enzyme, in Ca2+ and in EDTA buffers. (B) Anisotropy measurements on the binding of either the specific or the non-specific 18-mer (20 nM) to BcgI in Ca2+ buffer.

8. Free DNA (2 µM) A) DNA + 8.66 μM BcgI B) 2 2 Residuals Residuals 0 0 -2 -2 0.6 0.30 0.5 0.25 0.4 0.20 Absorbance at 539 nm Absorbance at 539 nm 0.3 0.15 0.2 0.10 0.1 0.05 0.0 6.85 6.90 6.95 7.00 7.05 7.10 5.85 5.90 5.95 6.00 6.05 6.10 Radius Radius Speed = 25,000 rpm Observed MW = 11,742 (expected 11,916) Analytical Ultracentrifugation of BcgI ± DNA AUC experiments were performed on BcgI in the presence of Hex-labelled 18-mers. Sedimentation was measured from the HEX label at 539 nm. These revealed that one A2B trimer binds to one DNA duplex (Fig. 7B). Thus, the free DNA was observed to have a MW of 11,742 (Fig. 7A), whereas the enzyme-DNA complex gave a MW of 182,112 (Fig. 7B). These molecular weights correspond, respectively, to that expected for the free DNA and for an (A2B)(18-mer) complex. Speed = 6,500 rpm Observed MW = 182,112 (expected 189,392) Figure 7. AUC data of absorbance at 539 nm against centrifugal radius, and residual fits of the free DNA (2 µM) (Fig. 7A) and (Fig. 7B) the DNA (2 µM) in the presence of BcgI (8.66 µM) at 20 °C.

9. A) B) 5 nM DNA, 3 nM BcgI 5 nM DNA, 1 nM BcgI 6 6 5 5 SC SC L1/L2 4 4 DNA (nM) DNA (nM) 3 3 2 2 FLL 1 1 L1/L2 FLL 0 0 0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240 Time (mins) Time (mins) Figure 8. Reactions at 37 ºC on two-site DNA at different BcgI concentrations, as indicated above each panel. Following the addition of the enzyme to the DNA, aliquots were removed, analysed by agarose gel electrophoresis and the concentrations of the following forms of the DNA determined: SC, supercoiled DNA ( ); FLL, full length linear DNA ( ); L1/L2;, doubly cut products ( ).The bottom panel shows the forms of the DNA that can be generated during the reaction: SC, OC (open circle DNA), FLL, and L1/L2. Titrations of BcgI on two-site DNA at low enzyme concentrations At low enzyme concentrations (Fig. 8A), reactions of BcgI on a plasmid with two BcgI sites gave as the main product the full length linear (FLL) DNA cleaved at only one site, with only a little cleaved at both sites. In contrast, at higher enzyme concentrations (Fig. 8B), the predominant product released was the DNA cleaved at both sites (L1 and L2). 2x OC FLL L1 & L2 SC

10. SC (5 nM) One-site DNA/BcgI titrations SC (8 nM) SC (16 nM) BcgI titrations under single-turnover conditions Under single-turnover conditions with enzyme in excess of the DNA, BcgI cleaved a plasmid with two BcgI sites in a monophasic reaction, in which all of the DNA was cleaved (Fig. 9A). This differs from the reactions with DNA in excess of the enzyme when only a fraction was cleaved (Fig. 8). On a plasmid with one BcgI site, both the rates and the extents of cleavage were less than those observed at the same enzyme concentration on the two-site DNA (Fig. 9B). A) B) Two-site DNA/BcgI titrations 6 6 5 5 4 4 3 3 SC (nM) SC (nM) 2 2 1 1 0 0 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 Time(mins) Time (mins) Figure 9. BcgI titrations on one-site and two-site DNA. Following the addition of enzyme (at the concentrations indicated) to 5 nM DNA, aliquots were removed from the reaction and analysed by agarose gel electrophoresis. The reactions were performed at 37 ºC in BcgI buffer.

11. Conclusions • AUC experiments suggest that BcgI is an A2B trimer in solution and when bound to DNA. The A2B trimer seems to bind a single duplex, as judged by fluorescence anisotropy. Hence, in order to act concertedly on a DNA with two sites, two trimers may need to bind to the separate sites and then interact with each other. • In reactions with [Enzyme] < [DNA], titrations of BcgI on a two-site DNA indicate that, at low enzyme concentrations, only a small fraction of the DNA is cleaved, mainly to the product cut at just one site. However, at higher enzyme concentrations, a larger fraction is cleaved and the predominant product is DNA cleaved at both sites. However, under [Enzyme] < [DNA] conditions, complete cleavage of the DNA is never observed. This indicates that BcgI acts stoichiometrically rather than catalytically, at a single site at low [Enzyme] and at both sites at higher [Enzyme]. • Monophasic reactions that reach completion are only observed on two-site DNA when [Enzyme] > [DNA]. However, on one-site DNA, the reaction is always slower than that on the two-site DNA and always yielded a lower extent of cleavage. • On one-site DNA, the reaction can occur in trans, as shown by “oligo-activation” experiments.

12. Future Work Experiments with plasmid substrates fail to reveal whether the product released is DNA with double-stranded breaks on one side or on both sides of the recognition site. It may be possible to determine whether the cleavage occurs on both sides by analysing samples from the reactions on both : low percentage agarose gels, to measure the large (kb) fragment high percentage polyacrylamide gels, to monitor the appearance of the 34 bp fragment. On oligonucleotide substrates, primer extension experiments may reveal whether BcgI cleaves on the side near to or distant from the primer. • REFERENCES • Kong, H., Roemer, S.E., Waite-Rees, P.A., Benner J, Wilson GG, and Nwankwo DO (1993) Characterization of BcgI, a new Kind of Restriction-Modification System. J. Biol. Chem., 269, 683-690. • Kong, H., (1998) Analyzing the functional organization of a novel restriction modification system, the BcgI system. J. Mol. Biol., 279, 823-32. • Kong, H., and Smith, C.L., (1997) Substrate DNA and cofactor regulate the activities of a multi-functional restriction-modification enzyme, BcgI. Nucleic. Acids. Res, 25, 3687-3692. ACKNOWLEDGEMENTS We would like to thanks New England Biolabs for providing us with the strain required for purification of the BcgI enzyme and the BBSRC for funding this project.