Isolation of temperature-sensitive mutants of the BamHI restriction endonuclease

Gene, 157 (1995) 303-310 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50

303

GENE 08608

Isolation of temperature-sensitive mutants of the BamHI restriction endonuclease (Restriction enzyme; hydroxylamine mutagenesis; heat/cold-sensitive mutant; conditional-lethal mutant; SOS response)

Alexey Fomenkov* and Shuang-yong Xu New England Biolabs Inc., Beverly, MA 01915, USA

Received by F. Barany: 16 August 1994; Revised/Accepted: 1 November/2 November 1994; Received at publishers: 14 November 1994

SUMMARY

Two heat-sensitive R.BamHI mutants, T 1571 and P 173L, and one cold-sensitive R.BamHI mutant, T 114I, were isolated after chemical mutagenesis of the bamhlR gene that codes for the restriction endonuclease BamHI (R.BamHI). The thermosensitivity of Tl14I, T157I and P173L is revealed by the 102-103 lower plating efficiency at the non-permissive temperature of strains bearing these alleles. The conditional-lethal phenotype can be rescued by introduction of the cognate bamhlM gene into the same cell. The mutant enzymes induce the SOS response in vivo and display reduced phage restriction activity. The P173L protein, when expressed at 30°C and purified, shows reduced thermostability at 65°C. T157I and P173L mutants yield different intermediates during partial trypsin digestion. The conditional-lethal BamHI mutants could be used to deliver in vivo DNA cleavage and for further isolation of relaxed-specificity mutants.

INTRODUCTION

More than 180 restriction endonucleases (ENases) are now known (Roberts and Macelis, 1993). The BamHI ENase was one of the first discovered and characterized (reviewed by Nardone and Chirikjian, 1987). BamHI cleaves the palindromic sequence 5'-GGATCC-3' in double-stranded DNA, generating a 4-base 5' extension. Correspondence to: Dr. S.-y. Xu, New England Biolabs Inc., 32 Tozer Road, Beverly MA 01915, USA. Tel. (1-508) 927-5054, ext. 286; Fax (1-508) 921-1350; e-mail: [email protected] *Present address: Department of Biology, Johns Hopkins University, Baltimore, MD 21218-2685, USA. Tel. (1-410) 516-7330.

Abbreviations: A, absorbance (lcm); aa, amino acid(s); BamHI, R.BamHI; 13Gal, 13-galactosidase;bp, base pair(s); BSA, bovine serum albumin; cfu, colony-forming unit(s); cs, cold-sensitive; DTT, dithiothreitol; ENase (R.), restriction endonuclease; hs, heat-sensitive; kb, kilobase(s) or 1000 bp; IPTG, isopropyl-[3-D-thiogalactopyranoside; MTase (M.), DNA methyltransferase; PA, polyacrylamide; PAGE, PA-gel electrophoresis; R, resistance/resistant; SDS, sodium dodecyl sulfate; ts, temperature-sensitive; wt, wild type; [], denotes plasmidcarrier state. SSDI 0378-1119(94)00819-1

The gene coding for BamHI, bamhlR, has been cloned, sequenced and expressed in E. coli (Brooks et al., 1989; 1991; Jack et al., 1991). The recombinant BamHI protein (24570 Da) has been purified to homogeneity, crystallized, and its crystal structure determined (Newman et al., 1994). Genetic and biochemical studies showed that residues D 94, E 111 and E 113 of BamHI are important for the catalytic function (Xu and Schildkraut, 1991, Dorner and Schildkraut, 1994). ENases are toxic to host cells because of their DNA cleavage activity. To avoid endonuclease attack on intracellular DNA, a cognate MTase modifies a particular base in the same site, rendering the target sequence refractory to ENase cleavage. However, a special class of EcoRI mutants have been isolated that damage DNA despite methylation of EcoRI sites; these mutants display reduced sequence specificity as evidenced in enhanced star cleavage activity (Heitman and Model, 1990). We would like to extend this method to BamHI, and accordingly set out to isolate temperature-sensitive (ts) mutants. In this paper we report the identification of three mutations in the bamhlR gene that result in ts activity.

304 RESULTS A N D D I S C U S S I O N

(a) Isolation of BamHI mutants The method for isolation of BamHI ts mutants was basically the same as described previously (Heitman et al., 1989; Xu and Schildkraut, 1991). We took advantage of the observation that constitutive expression of BamHI from a Ptac promoter construct is lethal to the host in the absence of expression of its cognate MTase. C--,T transition mutations were introduced into the bamhIR gene by hydroxylamine mutagenesis. We used direct plating to screen BamHI ts mutants. The mutagenized plasmid DNA was used to transform E. coli strain ER2267. By screening over 800 colonies at 42°C and retesting at 30°C, we obtained 30 ts BamHI mutants. For hs ENase, the nonpermissive temperature of cells is 30°C (cell is not viable because the ENase is active) and the cell permissive temperature is 42°C (cell is viable since the ENase is not active); for cold-sensitive (cs) enzyme, the non-permissive temperature of cells is 42°C and the cell permissive temperature is 30°C. Only seven isolates were 'tight' ts mutants, i.e., cells are viable at 42°C but grow poorly at 30°C. The other 23 mutants were 'leaky' in that cells only show partially retarded growth at 30°C. The plating efficiency of cells carrying seven 'tight' ts mutants was measured and found to be at least 103-fold lower at 30°C compared to 42°C (Table I, lines 5 and 7, only two unique mutants are shown). In contrast, 'leaky' ts mutants displayed less than a five fold reduction in plating efficiency or formed small colonies at normal efficiency at 30°C. The mutations responsible for the 'leaky' ts phenotype were not further characterized. DNA sequencing of the entire bamhlR gene (642 bp) in the

seven remaining 'tight' mutants revealed that six mutants had the same C ~ T transition mutation in codon 173, converting Pro 173 to Leu (designated P173L). The seventh mutant had a C---,T transition mutation in codon 157 (T 157I). T 157I has previously been isolated by virtue of its reduced activity (Xu and Schildkraut, 1991). By selecting survivors at 30°C and replica-plating at 42°C, we also isolated two cs BamHI mutants. Cells were viable at 30°C, but grow poorly at 42°C (the mutant BamHI protein has more cell-killing activity at 30°C than at 42°C in vivo). Sequencing revealed that both mutants have a C ~ T transition at codon 114 (Tll4I). T l 1 4 ! has also been isolated before as a mutant with reduced activity (Xu and Schildkraut, 1991). Cells carrying the cs BamHI mutant showed a 250-fold decrease in plating efficiency at 42°C. A second mutagenesis attempt using a mutD strain identified a ts mutant with the same C - , T transition (P173L). EcoRI ts mutants have been isolated before; in contrast to BamHI ts mutations, aa substitutions resulting in EcoRI ts were located in a-helix (M255I, T261I and L263F in ~5) or in 13-sheets (R56Q in I~l) in the EcoRI crystal structure (Heitman et al., 1989; Kim et al., 1990). The EcoRI ts mutants have been used to deliver DNA damage in vivo. It was found that the major component for the repair of DNA breaks is DNA ligase (Heitman et al., 1989). Fig. 1 shows the positions of aa substitutions in the BamHI crystal structure (the side chains of T114, T157 and P173 are shown in bold). The Tl14 residue is located next to the putative catalytic center formed by D94, E111 and E113. T l 1 4 is also near the dimer interface formed by a-helices 4 and 6 (a-helix 4: aa 117-132; a-helix 6, aa 159-169). T157 is positioned near the putative DNA

TABLE I Effect of BamHI ts mutants on cell plating at permissive and nonpermissive temperatures on strains LD2264 and ER2267 a

BamHI allele

bamhlM

cfub

Permissive/ non-permissivec

30°C LD2264 bamhlR + LD2264[pBR322] ER2267[pBR322] LD2264[P173L] ER2267[P173L] LD2264[T157I] ER2267[T157I] LD2264ET114I] ER2267[Tl14I]

+ + + + + -

6.0 × 8.6 x 7.5 × 7.1 x 7.5 × 6.0 × 1.0 × 4.8 × 5.0 ×

42°C 108 l0 s 10s 108 102 l0 s 102 108 106

4.8 × 5.9 x 6.2 × 5.5 × 1.0 × 6.0 × 3.0 × 6.5 × 2.0 ×

108 108 108 108 106 108 105 108 104

0.80 0.69 0.83 0.77 1.33 × 103 1.00 3.00 × 103 0.74 2.50 × 102

a ER2267 {endA1 thi-1 supE44 e14-(McrA )A(mcrC-mrr)l14::ISlOA(argF-lac)U169 recA1 [F'proA+B+laclqA(lacZ)M15 zzf::mini-TnlO (KmR)]} (New England Biolabs catalog, 1993/94), LD2264 is isogenic with ER2267 except it carries a prophage L (Km R M . B a m H I ÷) and F' with Tc R (Dorner and Schildkraut, 1994). b cfu=colony-forming units. The numbers are average of three independent experiments. ° The ratio of permissive/nonpermissive=cfu (42°C)/cfu (30°C) for P173L and T157I. The ratio of permissive/nonpermissive= cfu (30°C)/cfu (42°C) for T1141.

305

Fig. 1. Crystal structure of BamHI protein as a dimer. The side chains of Tl14, T157 and P173 residues are indicated in bold. There is a large cleft (about 20 A wide and 15 ~, deep) for binding B-form DNA (Newman et al., 1994). binding cleft. Thus, aa substitutions at these two positions may perturb local structure critical for DNA binding and catalytic function. The aa substitutions at positions 114, 157 and 173 could also indirectly affect BamHI dimer interactions.

restriction activity is slightly temperature-sensitive. Restriction by the hs mutants T157I and P173L increased 3-4-fold at 30°C, consistent with greater activity at this temperature (Table II). The level of phage restriction increased 2-fold for the cs enzyme T l l 4 I at 42°C (Table II).

(b) Plating efficiency in the presence of the bamhlM gene If the BamHI mutant protein is responsible for the reduced plating efficiency, then introduction of the bamhlM gene should abolish the conditional-lethal phenotype. A second plasmid containing the bamhlM gene, pADE15 (M.BamHI +) was introduced into the cells that carry BamHI ts alleles. The plating efficiency in the presence of the bamhlM gene is shown in Table I (lines 4, 6 and 8). BamHI ts mutants are rescued by M ' B a m H I at the non-permissive temperature, which indicates that the cleavage specificity of the ts mutants is unaltered.

(c) Restriction of infecting ~ phages Although the BamHI ts mutants have reduced cleavage activity, they may still restrict phages. To test this possibility, cells carrying wt BamHI or the mutant endonucleases were infected with ~vir phages. The level of phage restriction by wt BamHI was almost unaffected when tested at 30°C and 42°C (1.5 × 10 -5 vs. 1.3 × 10-5). The overall phage restriction activity by the mutant enzymes dropped over three order of magnitude. The residual

(d) SOS induction by the mutant enzymes It has been shown that DNA double-strand breaks and single-strand nicks induce SOS response in E. coli (Panayotatos and Fontaine, 1985; Heitman et al., 1989). The level of SOS induction can be measured in strains that carry SOS-inducible loci fused to lacZ. To measure SOS induction by BamHI ts mutant, the plasmid carrying the P173L allele was introduced into a dinDl::lacZ fusion strain ER1992 (Fomenkov et al., 1994). When ER1992[pAEK-P173L] cells were incubated at 42°C, there was a 7-fold increase in ]3-galactosidase ([3Gal) activity compared with the background level (35 units vs. 5 units), reflecting residual activity of the mutant enzyme. After the incubation temperature was shifted to 30°C (non-permissive temperature) 13Gal activity decreased with time of incubation (15 units, 3 h after shift). We believe that after the temperature shift most cells were killed due to excessive DNA damage and therefore ]3Gal activity dropped to a lower level. T l l 4 I and T157I also have residual activity in vivo and induce the SOS

306 TABLE II Restriction of kvir by BamHI ts mutants BamHI allele

30°C

37°C

42°C

42°C/30°C

30°C/42°C

LD2264 [pBR322]a

0.9 b

LD2264[bamhlR +]

1.5 x 10-5 8.1 x 10 2 2.4 x 10-2 1.1 x 10-2

1.0 2.2 x 10-5 5.0 x 10 2 5.0 x 10-2 1.4 x 10-2

1.1 1.3 x 10-5 3.8 x 10 2 9.6 x 10-2 3.0 x 10-2

1.2 0.9

0.9 1.2 2.1

LD2264[Tl14I] LD2264[T157I] LD2264[P173L]

4.0 2.7

Phage )~was plated as described (Arber et al., 1983). LD2264 carries the bamhlM ÷ gene in a )~prophage. LD2264 cells carrying Tl14, T157I and P173L were first grown at the cell permissive temperature overnight and then diluted 100-fold into fresh tryptone broth plus maltose. Cells were grown at the permissivetemperature to 108 cfu/ml before phage infection. Phage infected cells were plated at 30, 37 and 42°C, respectively. b Values represent the ratio of phage titer on strain LD2264 bearing the indicated BamHI alleles divided by the titer on LD2264 at 37°C (e.g.,)~vir titer on LD2264 (L-M.BamHI +) is 2.6 x 10 9 at 37°C; )~vir titer on LD2264 0~-M"BamHI ÷, pAEKI4-R.BamHI +) at 42°C is 3.5 x 10 4, thus the level of phage restriction activity at 42°C by wt BamHI is 3.5 x 104/2.9 x 10 9 = 1.3 x 10-s. The phage restriction activities were derived from one experiment in triplicate. response at the cell permissive temperatures (2-5-fold increase in 13Gal activity). Introduction of the b a m h l M gene into ER1992 cells alleviates SOS induction by the mutant enzymes.

(e) Mutant BamHI activity in vitro and protein levels in cell extracts We interpret the decrease in colony-forming units (cfu) at the non-permissive temperatures to result from intracellular D N A damage in vivo and viability at the permissive temperature to result from enzyme inactivation. Accordingly, we wished to determine at what level inactivation occurred. Cell extracts were prepared from cells grown at 30°C and assayed for D N A cleavage activity at 42 ° and 30°C. Cell extracts of T157I and P173L prepared from 30°C cell cultures exhibits no major differences in D N A cleavage activity at 30°C and 42°C (data not shown). We observed the same activity for the T l 1 4 I enzyme assayed at 30°C or 42°C in the cell extract prepared from 42°C cells (data not shown). Thus, the hs phenotype of T157I and P173L and the cs phenotype of T1141 are only manifested in vivo. However, cell extracts of P I 7 3 L prepared from 30°C cell cultures showed a 4-8-fold higher D N A cleavage activity than cell extracts prepared from cells grown at 42°C (Fig. 2G and H). A 1:8 dilution of a 30°C cell extract gave a similar pattern to the undiluted 42°C cell extracts. T157I also shows a 2 to 4-fold higher D N A cleavage activity from the 30°C extracts as compared to the 42°C extracts (Fig. 2E and F). N o apparent differences in D N A cleavage activity were detected for wt B a m H I and T1141 at the two temperatures (Fig. 2, compare A and B; C and D). To further investigate the difference in activity at the two temperatures, Western blots were performed to assay protein levels in the soluble fraction of the cell extract using polyclonal rabbit a n t i - B a m H I serum. Fig. 3A shows that wt B a m H I protein reached its highest level at 6 h of I P T G induction. The protein levels are comparable at 30°C and

42°C. T l I 4 I (Fig. 3B) shows a similar result to wt B a m H I . The level of T157I protein is about 2-fold less at 42°C than at 30°C after I P T G induction for 6 h (Fig. 3C, lanes 4 and 8). A faint band of about 23 kDa, possibly a degraded form of T157I, was also detected (Fig. 3C, lanes 4, 6, 7 and 8). The amount of P173L protein at 42°C is about 6-fold lower than at 30°C (Fig. 3D, compare lanes 4 and 8). A Western blot of the total cellular proteins showed that the amount of P173L protein is also lower at 42°C (Fig. 4, compare 6 h of induction at 30°C and 42°C). At 30°C, the level of P173L protein increases with time of I P T G induction ( 1, 3 and 6 h). At 42°C, the level of P173L protein increases during the first hour of induction but decreases thereafter, possibly reflecting degradation.

(f) In vitro activity of the purified mutant ENases To study the in vitro activity of the mutant ENases, T114I, T157I and P173L proteins were purified to homogeneity from cells with the MTase, grown at the temperature at which the enzyme is active. When ENase activities of these three proteins were assayed at 30°C and 42°C, there was no more than 2-fold difference in cleavage activity between the two temperatures (data not shown). However, the hs mutant protein P173L is more readily inactivated by heat denaturation at 65°C (see section h).

(g) Trypsin digestion of the mutant proteins In order to reveal any overall structural changes of the mutant proteins, purified T l l 4 I , T157I and P173L proteins were subjected to partial trypsin digestion at 30°C and 42°C. The trypsin-digested fragments were detected by a n t i - B a m H I serum (Fig. 5). Two to three extra bands in trypsin-digested T157I and P173L appeared (Fig. 5, P173L digested at 30°C, lane 8; T157I digested at 42°C, lane 16; P173L digested at 42°C, lane 18, the extra bands indicated by a dot). The differences in protease accessibil-

307

A. wt BamHI, 30°C 1 2

1

1 4

1 8

1 16

C. T1141, 30°C 1 2

1

1 4

1 8

D. T1141, 42°C

1 16

G. P173L, 30°C 1

2

1 4

B. wt BamHI, 42°C

1 1 1 1 1 32 64 128 256 512

E. T1571,30°C 1

1 2

1 4

1 8

F. T1571,42°C

1 16

H. P173L, 42°C

1 1 1 1 1 1 1 8 16 32 64 128 256 512

Fig. 2. DNA cleavage activity of wt BamHI (A and B), mutant enzymes Tl14I (C and D), T157I (E and F) and P173L (G and H) in cell extracts prepared from cells cultured at 30°C or 42°C. All cleavage reactions were performed under standard BamHI digestion conditions at 37°C. Dilution factor is indicated above each lane. Same dilution factors were applied to A and B, C and D, E and F, G and H, respectively. To make a cell extract, a 40-ml culture of cells carrying pAEK14 (BamHI ÷) or mutant derivatives were grown at 30°C or 42°C to a Asso .m of 0.5. IPTG was added to 0.4 m M final concentration and induction continued for 1, 3 or 6 h. Induced or uninduced cells were chilled on ice, centrifuged and resuspended in 1 ml of sonification buffer (10 m M Tris.HC1, pH 7.8/2 m M EDTA/10 m M 13-mercaptoethanol). Cell lysis was completed by addition of lysozyme and sonication. Cell debris was removed by centrifugation and the resulting supernatant mixed with an equal volume of 100% glycerol. Phenylmethylsulfonylfluoride (PMSF) was added to 1 m M final concentration. Samples were stored at - 2 0 ° C . DNA cleavage products were resolved in 0.8% agarose gel.

ity suggest that there may be some conformational alteration of T157I and P173L proteins due to aa substitutions in the turns.

(h) Heat inactivation of the mutant proteins To examine the heat stability of the mutant proteins, the mutant proteins were purified to homogeneity and heated at 65°C for 2, 5, 10, 15 and 20 min. The heated proteins were diluted and used to cleave XmnI-linearized pUC19 DNA at 37°C. Fig. 6 shows the remaining activity of wt BamHI and the mutant proteins after heat inactivation. Before heat inactivation, wt BamHI shows a specific activity of 3 × 105 units/rag protein on linearized pUC19 DNA substrate. After 20 min incubation at 65°C, it retains about 12.5% of its activity. Purified T1141 protein shows a specific activity of 2 × 104 units/mg. T1141 displays about 12.5% of its activity after 20 rain incubation at 65°C. T157I protein has a specific activity of 2 x 103

units/mg. The low specific activity of T157I is due to the loss of activity during protein purification and storage at -20°C, since in cell extracts T157I activity was comparable with Tl14I activity (see Fig. 2). T157I lost all the remaining activity after 20 min heat inactivation. P173L protein shows a specific activity of 1 x 105 units/mg. Unlike wt BamHI, P173L was completely inactivated after 20 rain incubation at 65°C. This heat inactivation experiment shows that: (i) the T1141 mutant is not significantly different from the wt; (ii) the T157I mutant activity is too low to decide that this enzyme is more rapidly denatured than the wt; (iii) P173L is extremely sensitive to heat denaturation in vitro.

(i) Conclusions (I) E. coli strains harboring hs BamHI variants T157I and P173L, and a cs variant Tl14I grow poorly at the non-permissive temperatures. All three aa substitutions

308

A. wt BamHI 30°C

B, Tl141 30°C

42°C

t

tf

0136

1

0 13

1

6 h

42°C II

I

o136

01

3 6 ~

h i~ ~ ~/i~ ~ i

kDa kDa

-36.5

-36.5 -26.7

"

-26.7

-20.0 -20.0

123

4

5678

9

1234

C. T1571 30°C I

36

42°C

30°C 1

0 136

r

h

5678

42°C II

0136

w

1 234

9

D. P173L

t[

01

5678

1

0 136

h

kDa

kDa

- 36.5

-36.5

-26.7

-26.7

- 20.0

-20.0

9

1 2 34

5 678

9

Fig. 3. Western blot of wt BamHI and of Tl14I, T157I and P173L mutant proteins in cell extracts (soluble fraction) prepared from cells cultured at 30°C or 42°C. The time of IPTG induction (0, 1, 3 and 6 h) is indicated above each lane. Lane 9, purified wt BamHI protein (24.6 kDa). Rabbit antiBamHl serum was obtained after the third injection of the purified BamHI protein. After 0.1% SDS-10-20% PAGE, proteins were transferred onto nitrocellulose membrane by electroblotting. The wt BamHI and mutant proteins were detected by rabbit anti-BamHI serum and anti-rabbit IgG conjugated with alkaline phosphatase. To estimate the amount of protein, Western blots were scanned in a Microtek scanmaker.

resulting in ts phenotype are located in the turns in the BamHI crystal structure. (2) Presence of M.BamHI in the same cell suppresses the conditional-lethal phenotype. (3) The mutant enzymes induce SOS response in vivo and display reduced phage restriction activity. (4) The mutant protein P173L is more rapidly inactivated at 65°C. (5) T157I and P173L proteins display reduced stability at 42°C in vivo under IPTG-induced condition.

(6) T157I a n d P173L proteins yield different intermediates in partial trypsin digestion.

ACKNOWLEDGEMENTS We t h a n k W i l l i a m E. Jack, Elisabeth A. Raleigh, Richard Roberts a n d Ira Schildkraut for critical comments, Sanjay K u m a r for help with Fig. 1, Aneel Aggarwal for p r o v i d i n g the B a m H I crystal structure coordinates, Joseph H e i t m a n for advice, a n d Elisabeth A.

309 125

P173L 30°C 42°C ~0 1 3 6Rio 1 3 61h

100 wt = 75 ..-&

T114I

.-= 50-

Pl731

i~~

kDa

....

--

36.5

--

26.7

--

25-

: -

0

5

20.0

Trypsin digestion at

-,,~

6""'b"........ ~ 0

.~

[]

0

0

15

20

10

T157I

25

Time (rain) Fig. 6. Heat inactivation of the BamHI mutant proteins at 65°C. 100 ng ofwt BamHI or mutant proteins (in 100 ~tl of l x BamHI buffer/150 m M NaC1/10 m M Tris.HC1, pH 7.5/10 m M MgCI2/1 m M DTT/0.1 mg per ml BSA) were heated at 65°C for 2, 5, 10, 15 and 20 min. 10-gl aliquots were taken at each time point and twofold serial dilutions were made (1:2 up to 1:512). The diluted aliquots were used to cleave XmnI-linearized pUC19 DNA at 37°C for 1 h. The BamHI mutant proteins were purified to homogeneity by DEAE-Sepharose chromatography, heparinSepharose chromatography and Mono-Q FPLC. Protein was quantified using the Bio-Rad Protein Assay. Cells carrying T157I and P173L alleles were grown and induced at 30°C. Cell culture of Tl14I was grown at 42°C. The mutant proteins were purified at 4~C and stored at - 2 0 ° C in 50% glycerol in a BamHI storage buffer.

Fig. 4. Western blot of PI73L BamHI mutant protein in total cell extract (omission of centrifugation step after lysis of ceils by lysozyme treatment and sonication). This total cell extract includes both soluble and insoluble fractions. The time of IPTG induction (0, 1, 3 and 6 h) is indicated above each lane.

A.

.... "~....

B.

30°C

Trypsin digestion at

42°C

Tl141 T1571 P173L WT

Tl141 T1571 P173L WT

Trypsin ~ -3 -2 " -3 -2 " -3 -2 " -3 - 2 . . . dflut~ons:lo 10 10 10 10 10 10 10 M

U

J

ii

li

il

I

1031()21()31()2ld3 1(32163ld2 U

kDa -

175.0

-

-

83.0 62.0 47.5

-

32.5

-

25.0

-

16.5

-

6.5

-

....

1

2

34

5

6 7 8

9

101

12 13 14 15 16 17 1 8 1 9

20

21 2 2

Fig. 5. Trypsin partial digestion of the BamHI mutant proteins at 30°C and 42°C. Trypsin (1 mg/ml) was diluted 1:100 and 1:1000 and used for the partial digestion of wt BamHI and the mutant proteins. M, prestained protein size marker. The additional bands found in partial digestion of T157I and P173L proteins were indicated by a dot. Trypsin (1 mg/ml) was prepared in 10 m M HCI. Ten ~tl of the diluted protease (10 -1, 10 -2 and 10 -3 dilutions) were mixed with 6 ~tg of protein in a 100 ~tl of reaction volume and incubated at 30°C or 42°C for 30 rain. The trypsin digestion was terminated by addition of 10 ~tl of trypsin inhibitor (1 mg/ml) and protein precipitated with acetone. The protease digested fragments were separated by electrophoresis in a 0.1% SDS-10-20% PA gradient gel, transferred to a nitrocellulose membrane and detected by anti-BamHI polyclonal antibodies.

310 R a l e i g h a n d L y d i a D o r n e r for p r o v i d i n g strains. T h i s w o r k w a s s u p p o r t e d by N e w E n g l a n d B i o l a b s .

REFERENCES Arber, W., Enquist, L., Hohn, B., Murray, N., and Murray, K.: Experimental methods for use with lambda. In: Hendrix, R.W., Roberts, J.W., Stahl, F.W. and Weisberg, R.A. (Eds.), Lambda II. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1983, pp. 433-458. Brooks, J.E., Benner, J.S., Heiter, D.F., Silber, K.R., Sznyter, L.A., JagerQuinton, T., Moran, L.S., Slatko, B.E., Wilson, G.G. and Nwankwo, D.O.: Cloning the BamHI restriction-modification system. Nucleic Acids Res. 17 (1989) 979 997. Brooks, J.E,, Nathan, P.D., Landry, D., Sznyter, L.A., Waite-Rees, P., Ires, C.C., Mazzola, L.M., Slatko, B.E. and Benner, J.S.: Characterization of the cloned BamHI system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts. Nucleic Acids Res. 19 (1991) 841-850. Dorner, L.F. and Schildkraut, I.: Direct selection of binding proficient/ catalytic deficient variants of BamHI endonuclease. Nucleic Acids Res. 22 (1994) 1068-1074.

Heitman, J. and Model, P.: Mutants of the EcoRI endonuclease with promiscuous substrate specificity implicate residues involved in substrate recognition. EMBO J. 9 (1990) 3369-3378. Heitman, L, Zinder, N.D. and Model, P.: Repair of the Escherichia coli chromosome after in vivo scission by the EcoRI endonuelease. Proc. Natl. Acad. Sci. USA 86 (1989) 2281-2285. Jack, W.E., Greenough, L., Dorner, L.F., Xu, S.-y., Strzelecka, T., Aggarwal, A.K. and Schildkraut, I.: Overexpression, purification, and crystallization of BamHI endonuclease. Nucleic Acids Res. 19 (1991) 1825-1829. Kim, Y., Grable, J.C., Love, R., Greene, P.J. and Rosenberg, J.M.: Refinement of EcoRI endonuclease crystal structure: a revised protein chain tracing. Science 249 (1990) 1307-1309. Nardone, G. and Chirikjian, J.G.: The enzyme of the BamH! restrictionmodification system. In: Chirikjian, J. (Eds.), Gene Amplification and Analysis, Vol. 5. Elsevier, New York, NY, 1987, pp. 147-184. Newman, M.N., Strzelecka, T., Dorner, L.F., Schildkraut, I. and Aggarwal, A.K.: Structure of restriction endonuclease BamHI and its relationship to EcoRI. Nature 368 (1994) 660-664. Panayotatos, N. and Fontaine, A.: An endonuclease specific for singlestranded DNA selectively damages the genomic DNA and induces the SOS response. J. Biol. Chem. 260 (1985) 3173-3177. Roberts, R.J. and Macelis, D.: REBASE-restriction enzymes and methylases. Nucleic Acids Res. 21 (1993) 3125-3137. Xu, S.-y. and Schildkraut, I.: Isolation of BamHI variants with reduced cleavage activity. J. Biol. Chem. 266 (1991) 4425-4429.