A method was developed to isolate nicking variants from a library of SapI expression clones possessing random mutations (outlined in Figure ). The first step of the method is transformation of the library into bacterial cells where the genomic DNA is not protected by DNA methylation of SapI sites. In this step, a stringent selection occurs since RecA+
strains are significantly more tolerant of single-strand nicks as compared to double-strand cuts (13
). In this genetic selection, survivors are expected to be expressing nicking variants, low activity variants and null variants. The surviving cells are pooled and the plasmid library is isolated after a short outgrowth period. The library plasmid was pSAPV6, which was engineered to possess one SapI substrate site within the polylinker just downstream of the sapIR
open reading frame (Figure A). Therefore, plasmid clones expressing nicking enzymes may contain a site-specific and strand-specific nick upon isolation from the bacterial cell. This nick is the enabling factor for the in vitro
An outline of the selection procedures to isolate SapI variants with either top- or bottom-strand nicking activity.
Initial selection procedure for plasmids possessing a site-specific, strand-specific nick
A plasmid library expressing SapI variants was constructed as detailed in the Materials and Methods. The pSAPV6 ligation mix was transformed into ER1992 cells by electroporation and plated on LB agar containing 30 μg/ml chloramphenicol and 80 μg/ml X-gal. ER1992 is a DNA damage indicator strain, which contains the dinD1::lacZ
gene fusion (18
). When cells are plated on media containing X-gal, the extent of blue color development is a measure of the SOS response. A first assumption was made that full-strength nicking variants might induce the SOS response in Escherichia coli
while variants possessing double-strand cleavage activity would result in cell lethality. ER1992 does not express the T7 RNA polymerase. Yet, a low level of sapIR
gene expression was evident as a mixture of colony phenotypes was observed. Medium-blue colonies reporting DNA damage were pooled in 2 lots (60 colonies each) and inoculated into LB plus Cam. The two 10 ml cultures were grown to saturation at 37°C and plasmid DNA was prepared.
The in vitro selection process comprises two parallel procedures designed to isolate and amplify the sapIR genes from plasmids carrying a site-specific nick at the SapI cleavage site (Figures and ). In addition to the SapI site, the expression vector pSAPV6 carries an N.BbvCI nicking site nearby (Figure A). Figure B displays the expected scenario for isolating the sapIR genes from plasmids containing a SapI top-strand nick. If the library contains any plasmids with a nick after 5′-GCTCTTCG-3′, then digestion with N.BbvCIA will create a linearized plasmid with a pre-defined 10-base cohesive end. Similarly in Figure C, plasmids containing a SapI bottom-strand nick (before 5′-CGACGAAGAGC-3′) may be linearized by digestion with N.BbvCIB thus forming a 4-base cohesive end. In both scenarios, a single-stranded (ss) adaptor is ligated and the sapIR genes of interest are amplified by PCR using the strategy displayed in Figure D. Single-stranded adaptor 296–334 (Figure B) was designed to anneal to the cohesive end produced by SapI top-strand nicking and N.BbvCIA bottom-strand nicking. After ligation of the adaptor, PCR amplification of genes possibly encoding top nicking variants was accomplished using primer 275–224 that anneals to the last 20 nt of adaptor 296–334. The upstream primer in both cases is T7 promoter primer 298–024 with the sequence 5′P-GGGAGATCTCGATCCCGCGAAATTAATACG-3′. Importantly, one additional step is required for the procedure designed to isolate genes encoding bottom-strand variants (Figure ). After ligation of ss adaptor 296–335 to the 4-base cohesive end (Figure C), a Klenow fill-in reaction is required to produce a complementary strand to serve as a template for PCR amplification. Therefore in the Nb.SapI procedure, reverse PCR primer 275–224 anneals to the strand created by the Klenow reaction and not to the ligated adaptor (as in the Nt.SapI procedure).
In the first application of the method, PCR products of the expected size (1493 bp) were observed for both top and bottom selection procedures (data not shown). Templates from mock ligations (where adaptor was not added) did not yield such PCR products. The PCR products were digested with BamHI, isolated by agarose gel electrophoresis and cloned back into pSAPV6 (prepared by SmaI/BamHI digestion and CIP treatment). After the selected gene libraries were ligated to pSAPV6, the ligation reactions were transformed into ER2744 [pBR322-SapIM1M2], a pre-modified expression host. The clone pBR322-SapIM1M2 expresses both SapI methylase genes (14
) to modify and protect the SapI recognition sites present within the host genome.
Forty-eight transformants (32 top and 16 bottom) were screened for the expression of SapI nicking variants (see Materials and Methods). Of the 32 ‘top-strand’ clones, none expressed a nicking variant. However, one of the 16 ‘bottom-strand’ clones (variant 33) displayed significant nicking activity as indicated by the conversion of supercoiled pUC19 to relaxed, open circle form (Figure A, lane 1). Variant 39 (Figure A, lane 7) produced some linearized pUC19 while all other lanes display typical levels of open, circular substrate produced by non-specific nicking activities present in E.coli extract.
Figure 3 (A) Initial isolation of Nb.SapI-1 (variant 33). Nicking activity is revealed by incubating cell extract with supercoiled pUC19; (sc), supercoiled. Lane M is a 1 kb DNA ladder where the prominent band is a 3 kb band. The nicked form of pUC19 migrates (more ...)
Characterization of variant 33
Sequencing the sapIR gene of clone 33 revealed mutations resulting in four amino acid substitutions: D34Y/I82V/P168L/R420I. Positions 34, 168 and 420 were individually randomized by site-directed mutagenesis to determine which of these substitutions was responsible for the nicking phenotype. Limited screening of substitutions at position 34 and 168 did not result in a variant possessing nicking activity. However, several nicking variants were produced by randomizing position 420 (Table ). Variants with pronounced nicking activity were sequenced to discover many allowed substitutions: Asn, Ser, Cys, Thr, Leu, Ile, Val, Ala and Gly. As all these nicking variants possessed some degree of double-strand cleavage activity, none were superior to selected variant 33 (data not shown).
Variant 33 was partially purified by conventional chromatography to allow for more thorough characterization. The final titer of the enzyme was 2000 U/ml, where 1 U is defined as the amount of enzyme required to give complete nicking of 1 μg pUC19 in 60 min at 37°C. Figure B displays the nicking/cleavage characteristics of purified variant 33 (also named Nb.SapI-1). As the enzyme level is increased, some double-strand cleavage activity becomes apparent. Various buffer conditions were employed to possibly eliminate this residual double-strand cleavage activity. The following NEB buffers (with distinguishing characteristics in parentheses) were tested at a 1× concentration: N.BstNBI (150 mM KCl), BamHI (150 mM NaCl), BAL-31 nuclease (120 mM NaCl), DpnII (pH 6.0), EcoRI (pH 7.5), λ exonuclease (67 mM glycine–KOH, pH 9.4), MwoI (150 mM NaCl, 50 mM Tris–HCl, pH 7.9), NruI (100 mM KCl), NsiI (pH 8.4), Sau3AI (pH 7.0), ScaI (pH 7.4), TaqI (pH 8.4) and NEB standard buffers 1, 2, 3 and 4. One unit of variant 33 (as defined in NEB buffer 4) was added to each reaction for 60 min at 37°C. None of the conditions allowed complete conversion of supercoiled pUC19 to open, circular form without some linearization (data not shown). Therefore, the reaction conditions recommended for wt SapI (1× NEB buffer 4) appear to be most optimal for nicking variant 33.
Variant 33 was expected to be a bottom-strand nicking enzyme as defined by the selection method. The strand specificity was confirmed with sequencing reactions using a nicked pUC19 product as the template (see Materials and Methods). Variant 33 was incubated with 2 μg pUC19 and the nicked product was isolated by agarose gel electrophoresis. The sequencing reaction using primer #S1224S terminated at the expected position 5′…GCTCTTCCGCTA-3′ corresponding to a SapI bottom-strand nick (Figure ). Note that the final adenine peak is false as it is added by the terminal transferase activity of Taq
DNA polymerase. Sequencing the opposite strand of the pUC19 product revealed no evidence for the occurrence of nicking on the top strand. Variant 33 was then renamed Nb.SapI-1. Nb is the proposed nomenclature for N
icking enzyme, b
ottom strand (2
Figure 4 Run-off sequencing to determine the nicking site of Nb.SapI-1 (variant 33). The substrate pUC19 was incubated with purified protein for 30 min at 37°C. The nicked circular DNA product was isolated by agarose gel electrophoresis and sequenced (more ...)
Improved selection procedure for SapI nicking variants
The inability to isolate a top-strand nicking variant in the first procedure was most likely due to the limited size of the initial library (only 120 clones were selected for the first library). Furthermore, it was later determined that genetic selection in ER1992 was not stringent enough to eliminate variants with low double-stranded cleavage activity. For example, a survivor of the selection in ER1992 (variant 39) produced considerable linearization of pUC19 (Figure A, lane 7). Ideally, the genetic selection should eliminate all enzymes with measurable double-strand cleavage activity. We chose to repeat the genetic selection step with a strain encoding the T7 RNA polymerase to increase constitutive SapI expression from pSAPV6. In addition, a DNA damage indicator strain was not employed in the improved process since the desired SapI clone would most likely not produce a significant SOS response. This conclusion was made after clone 33 was transformed back into ER1992 to assess blue color development. The blue phenotype produced by variant 33 was very similar to the color of ER1992 transformed with empty vector. Therefore, a pure nicking variant recognizing a 7 bp sequence was expected to escape detection by the SOS indicator system of ER1992.
The same mutagenized sapIR gene library was ligated into pSAPV6 and transformed into ER2848 [ER2744 (lacIq)] by electroporation. Approximately 600 survivors were pooled into 500 ml LB plus Cam. The culture was grown to late log phase at 37°C, harvested by centrifugation and plasmid DNA was prepared by Qiagen® Maxiprep. The in vitro selection process was improved as well, by an agarose gel isolation of the slowly migrating DNA present in the pooled plasmid library. The slowly migrating plasmid DNA (comprising the nicked and the dimer forms) was excised from the gel and purified by adsorption to silica. This step eliminated most of the supercoiled plasmid DNA and enriched the library for nicked expression clones. The adaptor ligation and PCR amplification were carried out as before except for one change in the top-strand procedure. The adaptor was added after heating the N.BbvCIA nicked DNA to 65°C to ensure melting of the 10-base cohesive end (Figure A and B). Again, PCR products of the expected size were produced and the sapIR genes of interest were ligated to pSAPV6. The ligation mix was transformed into ER2744 [pBR322-SapM1M2] by electroporation.
Forty-eight ‘top strand’ transformants were analyzed for nicking activity. Of the 48 cell extracts, 5 were positive for significant nicking activity (Figure A displays the results of variants 1–19). All five positive clones (2
) were sequenced to determine the responsible amino acid substitutions. The two most active nicking variants (11 and 15) contained three substitutions in common: K80E, E250K and K273R. Variant 11 carried the additional substitutions Q81R, T109I and L193F and variant 15 carried the additional substitution I414T. Site-directed PCR mutagenesis was conducted to determine which of the common substitutions might be responsible for the nicking phenotype. In this study, both single variants E250K and K273R produced an exclusively top-strand nicked pUC19 product while variant K80E was negative for nicking activity. Mutations present in other selected clones led to the findings that substitutions Q240R and G271R will each independently produce top-strand nicking (see Table ). The four top-strand nicking variants were named Nt.SapI-1 (E250K), Nt.SapI-2 (K273R), Nt.SapI-3 (Q240R) and Nt.SapI-4 (G271R). Comparative analysis indicated that substitution E250K results in the most active top-strand SapI nicking variant. The improved selection process did not yield additional Nb.SapI variants. However, the fraction of desired clones from the improved top-strand procedure was 5 of 48 (10%) compared to 0 of 48 from the initial procedure.
Characterization of Nt.SapI-1 (E250K)
The strand specificity of variant E250K was confirmed by incubating pUC19 with purified protein followed by sequencing both strands of the nicked product (see Materials and Methods). The sequencing results are shown in Figure . The sequencing reaction using primer 266–113 terminated at 5′…GGAAGCA-3′. This truncated sequence is the product of using a nicked top strand as template. Top-strand nicking occurs between the first and second nucleotide downstream of the SapI recognition sequence. Note that the final adenine peak is false as it is added by the terminal transferase activity of Taq
DNA polymerase. The activity of purified Nt.SapI-1 (E250K) was assayed to investigate the characteristics of double-stranded cleavage. When 8 U of enzyme were incubated with 1 μg of pUC19 for 60 min at 37°C, there was no detectable linearization of pUC19 (Figure B). The specific activity of variant E250K is estimated to be higher than that of wt SapI. This feature has been documented previously in the creation of the nicking enzyme N.AlwI where the specific activity is 20-fold higher than wt AlwI (7
). Despite the high specific activity of variant E250K, the DNA damage indicator strain ER1992 did not report the induction of SOS when this clone was introduced.
Figure 6 Run-off sequencing to determine the nicking site of Nt.SapI-1 (E250K). The substrate pUC19 was incubated with purified protein for 30 min at 37°C. The nicked circular DNA product was isolated by agarose gel electrophoresis and sequenced using (more ...)
Figure 5 (A) Isolation of Nt.SapI variants containing the substitution E250K. Nicking activity is revealed by incubating cell extract with supercoiled pUC19. Variants #11 and 15 (lanes 11 and 15) carry the common substitutions K80E, E250K and K273R. All (more ...)
Nicking characteristics of wt SapI
Wild-type SapI was purified using the IMPACT-CN purification system (see Materials and Methods). The final enzyme concentration was adjusted to 2000 U/ml, which corresponds to 0.04 mg/ml or 800 nM monomer. A previous study by Bath et al
) evaluated the cleavage characteristics of SapI on substrates containing one or two sites. The conclusion was that SapI cleaves each site within each substrate at similar rates. This suggests that SapI does not need to interact with two recognition sites to accomplish DNA cleavage, in contrast to FokI, e.g. (20
). Furthermore, Bath et al
) concluded that SapI did not generate appreciable amounts of nicked product when 4.8 nM (12 U/ml) enzyme was incubated with 5 nM plasmid substrate. The nicking characteristics of wt SapI were re-evaluated in this study using a lower ratio of enzyme:substrate. In this study, 1 nM SapI was incubated with 8 nM supercoiled pUC19 at 37°C in 1× NEB buffer 4 plus 0.1 mg/ml BSA and aliquots were taken every 5 min throughout a 40 min reaction. The results of this time course reaction are displayed in Figure A. The first lane is an aliquot taken before enzyme addition to show the inherent amount of nicked substrate. As the reaction proceeds, linear product and nicked product both increase and then appear to become constant. This phenomenon was observed for enzyme concentrations from 0.1 to 1.0 nM (data not shown). Complete conversion to linear form within 40 min was only observed when the enzyme:substrate ratio was ≥1:2 (data not shown).
Figure 7 (A) DNA nicking/cleavage characteristics of wt SapI. SapI at a concentration of 1 nM was incubated with 8 nM pUC19 in a 200 μl reaction incubated at 37°C for 40 min. Aliquots of 20 μl were withdrawn every 5 min and added (more ...)
The nicked product observed in Figure A was isolated from the gel in order to analyze the nicks generated at the SapI cleavage site. The strand specificity was assessed by a second incubation with a 16-fold unit excess of wt SapI, Nt.SapI-1 or Nb.SapI-1 (Figure B). The results indicate that the nicks generated by wt SapI are not strand specific. Incubation of the ‘pre-nicked’ substrate with Nt.SapI or Nb.SapI allows the following conclusion, which is based on the amount of nicked substrate that is resistant to linearization. Approximately 70% of the nicks are on the bottom strand and 30% are on the top strand. This finding is corroborated by the results obtained from run-off sequencing reactions. Figure C displays the sequencing results of using the top strand as template and Figure D displays the results of using the bottom strand as template. In both sequence traces, the presence of a mixed peak indicates a mixture of templates where a fraction is nicked. Although the trace data is not quantitative, the more severe drop off of peak height in Figure D indicates that bottom-strand nicking is more prevalent than top-strand nicking. Finally, incubating the pre-nicked substrate with an excess of wt SapI (Figure B, lane 2) verified that the SapI recognition/cleavage site had not been damaged during the first digestion.