The generation of a single nick per plasmid molecule using restriction endonucleases with multiple recognition sites

63

Gene, 29 (1984) 63-68 Elsevier GENE 1017

The generation of a single nick per plasmid molecule using restriction endonucleases recognition sites

with multiple

(Recombinant DNA; ethidium bromide; open circle (Form II) DNA; single-strand cleavage; site-directed mutagenesis)

Balazs J. Kovacs, Stephen P. Gregory and Peter H.W. Butterworth* Department of Biochemistry, UniversityCollege London, Cower Street, London WCIE 6BT, (U.K.) Tel. 01-387-7050. ext. 299 (Received February lOth, 1984) (Revision received March 13th, 1984) (Accepted March 15th, 1984)

SUMMARY

Some restriction endonucleases generate a single-stranded nick at their recognition sequences in the presence of ethidium bromide (EtBr). This nick can then be extended to a single-stranded gap in which mutations can be introduced by a variety of techniques. To date, the templates used in these studies have largely contained a single recognition site for a given enzyme. Therefore, we have extended these studies to twelve enzymes for which multiple recognition sites exist in the template and show that, under appropriate conditions, one single-stranded nick is introduced per plasmid molecule.

INTRODUCTION

All methods for site-directed mutagenesis require some means of attacking a preselected DNA sequence. This can be achieved using synthetic oligonucleotides of defined sequence (Smith and Gillam, 198 1). Alternatively, use may be made of the sequen-

* To whom reprint requests should be addressed. Abbreviations: bp, base pairs; EtBr, ethidium bromide; Form I DNA, supercoiled circular DNA; Form II DNA, relaxed circular DNA; Form III DNA, linear DNA. 0378-l 119/84/%03.00 0 1984 Elsevier Science Publishers

ce-specific nature of DNA cleavage by restriction endonucleases. Under appropriate conditions and in the presence of the DNA-intercalating agent EtBr, certain restriction enzymes have been shown to nick DNA in one strand rather than cutting both strands of templates containing unique recognition sites (see e.g. Parker et al., 1977; Osterlund et al., 1982). There are a variety of techniques one might then adopt to produce mutations in the immediate vicinity of this nick (Shortle et al., 198 1). To extend the potential of this approach, we have now shown that certain enzymes possessing multiple recognition sites can insert a single nick into supercoiled DNA under appropriate conditions.

64

MATERIALS

AND

precipitation

METHODS

and the 5’-ends

polynucleotide (a) Templates

Gilbert

used

The recombinant containing

pTPG-7,

a fragment

used in all experiments

a 34Wbp

of trout genomic

plasmid DNA, was

labelled.

In the latter,

Sherratt,

the HaeII-B

fragment

by ethanol

precipitation

restriction

endonuclease

polyacrylamide/8.3

and cleaved with a second prior

inhibition

of

duced into a supercoiled

pH 8.0;

a unique restriction

MgCl;

100 pg/ml gelatin),

7 mM

a variable

2-mercaptoethanol;

concentration

and between

of EtBr

6 and 10 units of

restriction endonuclease. Reactions were incubated for 4-5 h at 25°C in the dark, and terminated by extraction

on 5””

gels.

with phenol/chloroform.

restriction

endonuclease

cleavage by EtBr has been well documented, for relatively few enzymes (Osterlund et al., particular, this approach has been used to conditions under which a single nick can

10 ~1 reactions contained 0.5 pg of supercoiled plasmid DNA in nicking buffer (20 mM Tris * HCl,

(see figure legends)

to analysis

M urea denaturing

RESULTS

The enzyme incubations

7 mM

and

(Twigg and

1980).

(b) Restriction

by Maxam

(1980). DNA samples were again recovered

the

3657-bp plasmid pAT153 was used which is the cloning vector pBR322 (see Sutcliffe, 1979, for selacking

were labelled with T4

as described

except those where the tem-

plate was radioactively

quence)

kinase

plasmid

site (Parker

but only 1982). In establish be intro-

DNA template

at

et al., 1977; Shortle

et al., 1982). Using the conversion plasmid DNA (Form I) to relaxed

of supercoiled circular DNA

(Form II), we have sought to determine whether this phenomenon can be extended to supercoiled DNA templates which possess multiple recognition sites. The data presented in Fig. 1 demonstrate how a variety of restriction endonucleases can be forced to

(c) Agarose gel electrophoresis

yield high levels of Form II DNA simply by changing (1) Native Ficoll-400

was

added

to 5”” and the samples

loaded onto a 1 T,, agarose gel in 0.089 M Tris base, 0.089 M boric acid, 0.002 M EDTA, pH 8.0. Electrophoresis was for 14-16 h at 30 V. (2) Denaturing Samples were adjusted to 5% Ficoll-400,

TABLE

I

The effect of EtBr on the percentage by a number

of restriction

of Form 11 DNA produced

endonucleases

from a Form I DNA

template

0.2 M

NaOH, 20 mM EDTA and then loaded onto a 19, agarose gel containing 30 mM NaCl and 2 mM EDTA. The running buffer was 30 mM NaOH, 2 mM EDTA and electrophoresis was for 14 h at 20 V. The gel was neutralised with 0.5 mM Tris . HCl, pH 7.0, prior to EtBr staining. Photographs of the stained gels were scanned using a Joyce-Loebl Chromoscan 3. (d) Labelling nicked DNA Nicked DNA samples (0.5 pg) were denatured at 95 “C for 5 min, chilled on ice and then incubated with bacterial alkaline phosphatase (BRL, 125 units) for 30 min at 65 “C. Following repeated phenol/chloroform extractions, DNA was recovered by ethanol

Enzyme

Number

of sites

per plasmid”

“” Form II DNA EtBr (pglml) 50

100

150

200





PLY411

I

PSf I

1

41

56

45

44

BglII

1

33

25

31

29

HaeII

3

33

28

30

32

.$haIII

5

34

30

35

33

‘~VUII

5

2

33

35

39

HinfI

6

29

22

17

24

.Alu I

9

13

84

67

33

HaeIII

13

Sou961

15

Hhol

17

Hp I I

18

* Plasmid pTPG-7 the template

used.


4

5

15

46

37

28

10

28

35

17

46

12

where pBR322 was

65

MboI 01

HpaYX

AluI

Fig. I. The effect of EtBr on the cleavage of template

(pTPG-7)

EtBr during

the incubation

EtBr. The number 7 indicate

one experimental

period:

of recognition

the presence

1234567

of DNA by twelve restriction

was used throughout.

endonucleases.

the concentration

of

atically by 50 pg/ml, yet at these low EtBr concentrations A/u1 and HpaII produce high yields of Form II DNA (80% or more, see Fig. 1 and Table I). The cleavage of pBR322 by PvuII is totally inhibited by 25 vg/ml EtBr whereas Sau961 produces high yields of Form II DNA at this concentration (not shown). Other enzymes require much higher levels of intercal-

Hinf I 234

AvaIL 01234

01234

All gels were of 1% agarose nicking

in nicking

and the same batch

of supercoiled

buffer containing

used is given in Table I. Lanes marked

50, 100, 150, 200, 250, 300 and 400 pg/ml EtBr, respectively,

EtBr in the reaction. The sensitivity to EtBr varies considerably between enzymes and is independent of the number of potential cleavage sites per plasmid. Some, such as Hue11 and AhaIII are inhibited dram-

23401

HaelIC

for 5 h at room temperature

for each enzyme

Haellt

23401

was run on each gel to check for non-specific

was incubated

sites in the template

of 0 (control),

parameter:

A control

0.5 pg template

01

I+,=

HhaI

01234

01234

234

AhalIt

I

DNA by 200 pg/ml

0, 1, 2, 3, 4, 5, 6 and

in the reaction

mix.

ating agent for the inhibition to be effective. For instance, the enzyme HhuI requires an EtBr concentration of > 300 pg/ml before it will yield > 70% Form II DNA. There are some enzymes, however, which seem incapable of generating high yields of Form II DNA under these conditions (see Fig. 1, BglII tracks). In these cases, the supercoil-to-linear conversion occurs without an appreciable lag phase at the open-circle state. Changing reaction conditions other than the EtBr concentration may facilitate the production of Form II DNA by such enzymes.

66

Table I contains the percentage of Form II DNA produced by a number of different restriction enzymes at different EtBr concentrations. Two enzymes which do not appear in this table deserve further mention for they can generate high yields (> 80 2) of Form II DNA in the absence of EtBr. Mb01 sites are methylated in E. coli by the dam methylase system and it is this presumably that inhibits double-strand cleavage resulting in open-circle production. This is inhibited by 50 pg/ml EtBr. NarI, which has a single site in the plasmid template used here, also generates high concentrations of Form II DNA in the absence of EtBr but this may be due to a marked difference in the rate of cleavage of some NarI sites (unpublished information from the supplier, New England Biolabs, Inc.). Having shown that enzymes with frequently occurring sites could generate high yields of Form II DNA, we wished to confirm that such DNA was indeed arising from the introduction of a single nick.

Fig. 2 shows an alkaline denaturing gel of nicked templates produced by four different restriction enzymes. In each case, only a linear and a closed-circle band are visible, suggesting that indeed there is only one strand cleavage event per plasmid. The apparent retardation of bands in the HhaI track may be due to the high concentration of EtBr used in the nicking reaction.

DEFG

Fig. 3. Ah11 nicks pATI Fig. 2. Agarose demonstrates

gel electrophoresis single-site

in the presence

cleavage

by denaturing

section

to completion AluI,

with Alul; lanes D-G,

lOOng/ml;

HhaI,

100 pg/ml, respectively. template

(see

with BumHI;

EtBr concentration

(shown

supercoiled

pTPG-7

for the production 300&ml;

HpaII,

treated

of EtBr (50ng/ml) MATERIALS

AND

endonuclease.

and nicks

conditions

control;

ison with labelled markers

(lane M). The size of labelled

with the

of Form II DNA: lOO~g/ml;

&I,

fragments

were sized by elec-

been assigned digestion

cleavage

is thought

Faint bands at 373,337,

to a low background

and do not correspond

at AluI sites.

pATI

is given in bp; the po-

sition of the A/u1 site from which each fragment

secondary

(see secon-

(lane A/H) and compar-

derived from HpaII-digested

derived is given in parentheses. 244 have

labelled

section d). Following

with HpaII, DNA fragments

under denaturing

site in the presence

with ALI in the presence

were radioactively

AND METHODS,

dary restriction trophoresis

at its recognition was treated

digested

See Table I for the number of sites in the

for each restriction

pATl53

enzymes

MATERIALS

lane C, pTPG-7

of EtBr. Plasmid

in Fig. 1)

of Form II DNA were analysed

c). Lane A, pTPG-7

linearised

conditions

restriction

of reactions

gel electrophoresis

lane B, pTPG-7 optimal

by certain

of EtBr. Aliquots

which yielded a high proportion METHODS,

under denaturing

to be

309 and

of incomplete

to selective

strand

67

A further experiment was performed with the &I-cleaved Form II DNA, this time using pAT153 as template, to confirm that this single nick was introduced at an &I site. The 5’-phosphate at the nick was replaced by radio-labelled phosphate and the plasmid subjected to secondary restriction with HpaII, prior to separating the fragments on a denaturing polyacrylamide gel. The sizes of the five prominently labelled products > 140 bp (Fig. 3) are as predicted from nicks inserted at five defined AluI sites in pAT153. Fragments shorter than 100 bp (not shown) also have sizes compatible with nicking at known A/u1 sites. Some variation in signal strength from the different AluI sites may be due to site preference which is known to occur in reactions carried out in the absence of EtBr (see Armstrong and Bauer, 1983). There is also evidence for selective cleavage of one of the two DNA strands at the recognition site. For example, pairs of equally labelled fragments of 476 and 146 bp and 226 and 266 bp should be derived from AZuI sites at map positions 15 and 1710 in pATl53. One of each pair is labelled more heavily than the other (see Fig. 3). It is not known whether the polynucleotide kinase used to end-label the nicks has any preference for particular Al&generated termini.

DISCUSSION

Gap misrepair mutagenesis is an extremely powerful tool to produce site-directed base substitutions in vitro (Shortle and Nathans, 1978; Shortle et al., 1982). Essential for such protocols is the generation of a nick at a known restriction site in the template. We have shown that this is possible not only with enzymes having unique restriction sites but also certain enzymes having multiple recognition sites in a template, thereby increasing the likelihood of occurrence of a potential nicking site within a DNA sequence of interest. In the case of AluI, it has been confirmed that this nick occurs at its recognition sequence. Although one should bear in mind that there may be some degree of site and/or strand preference, it should be possible to attack the adjacent base sequence, on either strand, from any chosen A/u1 site. Using the appropriate exonuclease, the nick can be extended into a gap of known dimen-

sions and the exposed single-stranded DNA is then susceptible to mutagenesis by a variety of techniques (reviewed by Shortle et al., 1981). This approach can also be used to expand a commonly occurring restriction sequence into a less frequent palindrome: for example, the AluI recognition sequence (AGCT) can be converted into Hind111 (AAGCTT), Sst I (GAGCTC) or PvuII (CAGCTG) sites, depending on the nature of the surrounding bases and the directionality of the gapping enzyme used.

ACKNOWLEDGEMENTS

The authors wish to thank Kate Rice for processing the DNA fragment size data. BJK is the holder of an SERC Research Studentship and the work was supported by grants from the SERC (GR/C/4417.4), the MRC (G8308275CB) and the Wellcome Trust.

REFERENCES Armstrong, K.A. and Bauer, W.R.: Site-dependent cleavage of pBR322 by restriction endonuclease HinfI. Nucl. Acids Res. 11 (1983) 4109-4126. Maxam, A.M. and Gilbert, W.: Sequencing end-labelled DNA with base-specific chemical cleavages, in Grossman, L. and Moldave, K. (Eds.), Methods in Enzymology, Vol. 65, Part I, Academic Press, New York, 1980, pp. 499-560. Osterhmd, M., Luthman, H., Nilsson, S.V. and Magnusson, G.: Ethidium-bromide-inhibited restriction endonucleases cleave one strand of circular DNA. Gene 20 (1982) 121-125. Parker, R.C., Watson, R.M. and Vinograd, J.: Mapping ofclosed circular DNAs by cleavage with restriction endonucleases and calibration by agarose gel electrophoresis. Proc. Natl. Acad. Sci. USA 74 (1977) 851-855. Shortle, D. and Nathans, D.: Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome. Proc. Natl. Acad. Sci. USA 75 (1978) 2170-2174. Shortle, D., DiMaio, D. and Nathans, D.: Directed mutagenesis. Annu. Rev. Genet. 15 (1981) 265-295. Shortle, D., Grisati, P., Benkovic, S.J. and Botstein, D.: Gap misrepair mutagenesis: efftcient site-directed induction of transition, transversion and frameshift mutations in vitro. Proc. Natl. Acad. Sci. USA 79 (1982) 1588-1592. Smith, M. and Gillam, S.: Constructed mutants using synthetic oligodeoxyribonucleotides as site-specific mutagens, in Set-

68 low, J.K. and Hollaender, A. (Ed%), Genetic Engineering, Vol. 3. Plenum, New York, 1981, pp. 1-32. Sutcliffe, J.G.: Complete nucleotide sequence of the E. coli plasmid pBR322. Cold Spring Harbor Symp. Quant. Biol. 43 (1979) 77-90.

Twigg, A.J. and Sherratt, D.: ‘frans-complementable cop) number mutants of plasmid ColEl. Nature 283 (1980) 216-218. Communicated by H.G. Zachau.