Proc. NatI. Acad. Sci. USA Vol. 74, No. 8, pp. 3213-3216, August 1977
Biochemistry
Cleavage specificity of the restriction endonuclease isolated from Ha emophil us gallinarum (Hga I)* (DNA sequencing/+X174 DNA/recognition sequences/cohesive ends)
NIGEL L. BROWNt AND MICHAEL SMITH1 Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England
Communicated by F. Sanger, May 16, 1977
The nucleotide sequences in the replicative ABSTRACT form (duplex) of 4X174 DNA around six sites cut by Hga I, a restriction endonuclease from Haemophilus galinarum, have been compared. The enzyme produces a staggered cleavage resulting in a pentanucleotide 5'-terminal extension. The sequences within and immediately surrounding the pentanucleotide cleavage site have no obvious relationship. However, the sequence 5'1--A-C-G-C-3' 3'-C-T-G-C-5' occurs five nucleotide pairs to the left of the cut in the upper strand and 10 nucleotide pairs to the left of the cut in the lower strand and, therefore, is believed to constitute the recognition site. This is a member of the class of restriction endonucleases in which recognition and cleavage sites lack 2-fold rotational symmetry. The method used to define the cleavage site is of
general applicability. An endonuclease from Haemophilus gallinarum, Hga I, (1, 2) is a typical class II restriction endonuclease (3) in that it cleaves susceptible DNA molecules into specific fragments and does not require S-adenosylmethionine or ATP as cofactors. Hga.I has been reported to cleave 4X174 replicative form (RF) DNA into 14 fragments (4) although the relative amounts of the fragments suggest that a number of nonstoichiometric digestion products may be present (ref. 4; unpublished results). The exact locations of six Hga I cleavage sites in the 4X174 DNA sequence have now been determined using a new method of characterization (5). The properties of the enzyme are analogous to those of Hph I (6) and Mbo II (7) (endonucleases of H. parahaemolyticus and Moraxella bovs, respectively), in that it apparently recognizes a specific asymmetrical pentanucleotide sequence. Hga I, however, is different in that the enzyme produces staggered cuts around an adjacent pentanucleotide, resulting in 5'-terminal extensions. The nearer of the two cuts is five base-pairs to one side of the proposed recognition sequence. Because there are no common features within the cleaved sequences, the resultant pentanucleotide 5'-terminal extensions do not have the potential to anneal in a random manner as is the case with terminal extensions produced in sequences with 2-fold rotational symmetry.
from H. aegyptius and Providencia stuartii, Hae III and Pst I, respectively, were prepared by methods similar to those described by Roberts et al. (8) and were stored in 50% glycerol at -20°. Escherichia coli DNA polymerase I was purchased from the Boehringer Corp. Ltd. and T4 DNA polymerase was donated by R. Kamen and A. R. Coulson. DNA Sequencing. The procedure used was essentially that of Sanger and Coulson (9). The qX174 am3 RF DNA and complementary strand DNA were gifts of C. A. Hutchison III. Mapping of Hga I Sites in 4X174 DNA. Some Hga I cleavage sites were mapped by comparison of (a) the fragment pattern obtained by double digestion of OX 174 RF DNA with Hga I and a second, previously mapped restriction endonuclease (refs. 10 and 11; see Fig. 3) with (b) the fragment patterns obtained with either enzyme alone. Location of Cuts in Hga I Cleavage Sites. For a given Hga I cleavage site the cuts made in each DNA strand by Hga I were separately aligned with the plus and minus gel pattern used to define the DNA sequence (9) in the region of the cuts. The principle of the method for aligning the cuts with the DNA sequence, as applied to a hypothetical symmetrical restriction endonuclease site, is shown in Fig. 1. The method is equally applicable to nonsymmetrical cleavage sites, and is based on a method described previously (5). DNA polymerase is used to extend a primer (usually a restriction fragment) and copy the DNA template to a very limited degree in the presence of the four deoxyribonucleoside 5'-triphosphates, one of which is a-32P-labeled. The triphosphates and the polymerase are then removed. These steps are common to the site determination and the plus and minus procedures (9). Two aliquots are then taken and T4 DNA polymerase I used to greatly extend the primer in the presence of the four unlabeled deoxyribonucleoside 5' -triphosphates (the "cold chase" step). The T4 DNA polymerase in one of the two reactions is inactivated with phenol (I). Treatment of this product with the datum restriction endonuclease (i.e., that defining the end of the primer) and with the restriction endonuclease under investigation produces a radioactive (copy) fragment which, when subjected to gel elecAbbreviations: Alu I, Hae III, Hga I, Hap II, HindII, Hph I, Hha I, Mbo II, Pst I, Taq I refer to restriction endonucleases isolated from Arthrobacter luteus, Haemophilus aegyptius, H. gallinarum, H. aphrophilus, H. influenzae Rd, H. parahaemolyticus, H. haemolyticus, Moraxella bovis, Providencia stuartfl, and Thermnus aquatcus, respectively (3, 17). RF DNA is the replicative (duplex) form of kX174 DNA. * The term restriction endonuclease is applied to Hga I even though it has not been shown to be involved in biological restriction. t Present address: Department of Biochemistry, University of Bristol, Medical School, University Walk, Bristol BS8 1TD, England. * Present address: Department of Biochemistry, Faculty of Medicine, University of British Columbia, Vancouver, B.C., Canada V6T iW5.
MATERIALS AND METHODS Enzymes. Samples of Hga I were donated by R. J. Roberts and G. B. Petersen. Endonuclease from H. haemolytcus, Hha I, was a gift of R. J. Roberts. That from H. aphrophilus, Hap II, was purchased from Miles Laboratories Ltd. Endonucleases The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
3213
3214
Proc. Nati. Acad. Sci. USA 74 (1977)
Biochemistry: Brown and Smith
Restriction fragment 5' primer annealed 31 to template
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3'-G-T-A-T-A-C-5' cleaved symmetrically to give (i) flush ends, (ii) tetranucleotide 5'terminal extensions, and (iii) tetranucleotide 3'-terminal extensions. Details are given in Materials and Methods.
trophoresis alongside the plus and minus pattern from the same primer fragment, defines the site of cleavage in the copy strand of the DNA. The second site location reaction (II), containing the active T4 DNA polymerase, is also treated with the datum restriction endonuclease and the enzyme under investigation. Initially the same fragment of radioactive DNA is generated as in the first experiment. However, the T4 DNA polymerase then acts on it to convert it to the same length as the nonradioactive template strand fragment. If there is a 5'-terminal extension, then the radioactive copy strand is extended, by repair synthesis to the same length as the template fragment. If there is a flush end, then there is no change because the four unlabeled deoxyribonucleoside 5'-triphosphates present repair any 3' exonucleolytic degradation. In the case of a 3'-terminal extension, this is removed by the exonuclease activity of the T4 polymerase (12) until the ends are flush. Analysis of the product of this reaction by gel electrophoresis alongside the plus and minus pattern indirectly, but quite specifically, defines the cut in the second DNA strand. Experimental details for the application of this method to Hga I sites are given in Fig. 2. RESULTS Mapping of Some Hga I Cleavage Sites in 4X174 RF DNA. A determination of the complete 4X174 DNA fragment
map for Hga I was not attempted because of the complex pattern of products, which included a number of persistent partial digestion products (unpublished results). Instead, double digestion of OX174 RF DNA with Hga I and Pst I was carried out. Pst I, which cleaves OX174 DNA at a single precisely characterized site (5), was found to cleave two fragments in an Hga I partial digest pattern. These two fragments, approximately 400 and 195 nucleotide pairs long (the longer of which must be incompletely digested with Hga I), were cleaved to give fragments approximately 235 and 160 nucleotide pairs long. This indicates that there is an Hga I site 160 nucleotide pairs to one side of the Pst I site and Hga I sites 235 and 35 (195 minus 160) nucleotide pairs to the other side of the Pst I site. The correct orientation of these sites relative to the restriction fragment map of OX 174 DNA (Fig. 3) was determined during experiments to precisely locate the cuts at each Hga I cleavage site. Additional Hga I cleavage sites were mapped by double digestion of 4X174 RF DNA with Hga I and Hae III. Inter alia, Hae III fragments 5 and 8 (ref 11; Fig. 3) were cleaved in the double digest, indicating the presence of Hga I cleavage sites within these fragments. These sites were precisely mapped during cut-location experiments. Sequences Surrounding Six Hga I Cleavage Sites in 4X174 am3 RF DNA. The specific location of the cut in each strand of the DNA was established for each site by the method described in Materials and Methods and the legend to Fig. 2. Illustrative experiments, together with experimental details, are shown in Fig. 2 and five sequences, containing six sites, are summarized in Fig. 4. The four sequences with only one cleavage site all have staggered cuts resulting in fragments with pentanucleotide 5'-terminal extensions. The striking characteristic of these cleavage sites is that the pentanucleotide extensions do not contain any common sequences, nor do the regions immediately adjacent. However, the pentanucleotide sequence 5'-G-A-C-G-C-3'
3'-C-T-G-C-G-5' occurs five nucleotide pairs to the left of three of the cleavage sites and, inverted, occurs five nucleotide pairs to the right of the fourth cleavage site. In the fifth sequence this pentanucleotide sequence occurs twice, separated by two nucleotide pairs (Fig. 4). Staggered cuts are found, the nearer of each pair of cuts being five nucleotides to the left of the sequence 5'-G-C-G-T-C-3' 3'-C-G-C-A-G-5' An apparent additional cut is reproducibly found in one strand of this sequence; its origin is not understood. Examination of the sequence of OX174 viral strand DNA (11) reveals that two contiguous sequences 5'-G-A-C-G-C-3' occur 157 nucleotides to the left of the Pst I site, near where a Hga I site was mapped in double-digestion experiments. DISCUSSION This paper describes the characterization of Hga I cleavage sites by a method that is generally applicable. The method has been shown (unpublished results) to work with enzymes known to give fragments with flush ends (Hindll, Alu I), 3'-terminal extensions (Hae II)., or 5'-terminal extensions (Hap II) and with one enzyme that does not cleave within a symmetrical sequence (Hph I) (see ref. 3). The method requires no prior knowledge
Biochemistry:
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Proc. Natl. Acad. Sci. USA 74 (1977)
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FIG. 2. Autoradiographs of plus and minus gel electrophoresis patterns for the determination of sequences cleaved by Hga I. Each plus and minus experiment was carried out as described by Sanger and Coulson (9), priming from a restriction enzyme cleavage site with complementary (nonviral) strand DNA as template. In addition, two aliquots (approximately 0.1 pmol of DNA) of the 32P-labeled random-length product from the first stage of the plus and minus reaction (9) were incubated for 30 min at 370 with T4 DNA polymerase (approximately 0.02 unit) and all four unlabeled deoxyribonucleoside 5'-triphosphates (0.4 mM) in buffer B (50 mM NaCl/6.6 mM MgC12/6.6 mM 2-mercaptoethanol/6.6 mM Tris.HCl, pH 7.4). One of the aliquots (I) was diluted with water (10 Ml) and treated with water-saturated phenol (25 gl). The aqueous layer was washed with ether (five times with 250 Ml) and the residual ether was removed by blowing air over the sample. The buffer concentration was adjusted to the concentration of buffer B. Both aliquots were then treated with Hga I and the same nuclease that was used to generate the plus and minus pattern. The two aliquots, and an aliquot of the product from the first stage of the plus and minus reaction (channel 0), were denatured and analyzed alongside the plus and minus experiment by electrophoresis through a 12% acrylamide gel containing 7 M urea/90 mM Tris-borate, pH 8.3/2.5 mM EDTA. The gel was then autoradiographed. The aliquot treated with phenol (channelI) defines the site of cleavage in the radioactive (copy) strand. The other aliquot (channel II) defines the cleavage site in the template strand. The autoradiographs shown were obtained after 5 days of exposure in order
is within a region of DNA whose nucleotide sequence can be determined (i.e., is within 100 nucleotides of a different restriction endonuclease cleavage site that can be used for primed synthesis). The method described here is modified from the version described in preliminary form (5) in that a "cold chase" step is used in the preparation of aliquot I (Fig. 1). This is to ensure that the DNA in aliquot I is a substrate for restriction enzymes such as Alu I and Hph I, which are not active on duplex DNA in the presence of extensive regions of single-stranded DNA (ref. 13, and unpublished results). In the experiments described here the cleavage sites are aligned with the plus and minus gel patterns obtained by the method of Sanger and Coulson (9). However, equivalent gel electrophoresis methods for DNA sequence determination (14) can be used for the sequence alignment.
Hga I resembles class II enzymes in its cofactor requirements (1,3), but like Hph 1 (6) and Mbo II (7) it cuts to one side of an asymmetrical pentanucleotide recognition sequence. The presumed recognition sequence is 5'-G-A-C-G-C-3' 3'-C-T-G-C-G-5' and the cleavage site is staggered, giving rise to pentanucleotide 5'-terminal extensions. The cut in the DNA strand containing the sequence 5'-G-A-C-G-C-3' is five nucleotides to the 3' (right) side of this sequence. The cut in the complementary strand is 10 nucleotides to the 5' (right) side of the sequence 3'-C-T-G-C-G-5'. Examination of a model of the DNA double helix shows that the cut sites lie on the same side of the helix, and the enzyme could function by aligning itself with the recognition site on the same side of the helix. Examination of the sequence of OX X174 am3 viral DNA (11) reveals that the sequences G-A-C-G-C and G-C-G-T-C occur 14 times. This is in agreement with the number of fragments produced on digestion of (X174 RF DNA with Hga I (4). However, a complete Hga I cleavage map has not been constructed due to the presence of persistent nonstoichiometric digestion products (ref. 4 and unpublished results). There may be several possible sources of such products. First, there are two sets of closely spaced recognition sequences, the pair separated by two nucleotides shown in Figs. 2 and 4, and a pair of contiguous recognition sites 157 nucleotides to the left of the Pst I site (nucleotides 5209-5219; ref. 11). Such closely spaced sites result in stable nonstoichiometric digestion products, as has been shown with Mbo 11 (7). In addition, there may be differences in the rates of cleavage of individual restriction sites (15), and this may be very marked for enzymes that do not cleave at their recognition sites. This has been seen in the digestion of OX174 RF DNA with Hph I (C. A. Hutchison III and M. Smith, unpublished results), and results in persistent partial digestion to show all the bands in the sequence. The bands in channels I and II, due to cleavage with Hga I, are therefore very intense. The correct alignment of these bands with the plus and minus sequence was obtained in 16- to 24-hr autoradiographs of the same gel. (A) Fragment 5 primer of the endonuclease from Arthrobacter luteus, Alu I, complementary strand template, cleaved at the Hha I 8b/4 site (Fig. 3). The detailed sequence is shown in Fig. 4 (ii). (B) fragment 3 primer of the endonuclease from Thermus aquaticus, Taq I, complementary strand template, cleaved at the Hap II 3/2 site (Fig. 3). The detailed sequence is shown in Fig. 4 (iv). (C) Hae III fragment 7 primer, complementary strand template, cleaved at the Hae III 7/5 site (Fig. 3). The detailed sequence is shown in Fig. 4 (v).
3216
Proc.. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Brown and Smith 0
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Biochemistry
Cleavage specificity of the restriction endonuclease isolated from Ha emophil us gallinarum (Hga I)* (DNA sequencing/+X174 DNA/recognition sequences/cohesive ends)
NIGEL L. BROWNt AND MICHAEL SMITH1 Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England
Communicated by F. Sanger, May 16, 1977
The nucleotide sequences in the replicative ABSTRACT form (duplex) of 4X174 DNA around six sites cut by Hga I, a restriction endonuclease from Haemophilus galinarum, have been compared. The enzyme produces a staggered cleavage resulting in a pentanucleotide 5'-terminal extension. The sequences within and immediately surrounding the pentanucleotide cleavage site have no obvious relationship. However, the sequence 5'1--A-C-G-C-3' 3'-C-T-G-C-5' occurs five nucleotide pairs to the left of the cut in the upper strand and 10 nucleotide pairs to the left of the cut in the lower strand and, therefore, is believed to constitute the recognition site. This is a member of the class of restriction endonucleases in which recognition and cleavage sites lack 2-fold rotational symmetry. The method used to define the cleavage site is of
general applicability. An endonuclease from Haemophilus gallinarum, Hga I, (1, 2) is a typical class II restriction endonuclease (3) in that it cleaves susceptible DNA molecules into specific fragments and does not require S-adenosylmethionine or ATP as cofactors. Hga.I has been reported to cleave 4X174 replicative form (RF) DNA into 14 fragments (4) although the relative amounts of the fragments suggest that a number of nonstoichiometric digestion products may be present (ref. 4; unpublished results). The exact locations of six Hga I cleavage sites in the 4X174 DNA sequence have now been determined using a new method of characterization (5). The properties of the enzyme are analogous to those of Hph I (6) and Mbo II (7) (endonucleases of H. parahaemolyticus and Moraxella bovs, respectively), in that it apparently recognizes a specific asymmetrical pentanucleotide sequence. Hga I, however, is different in that the enzyme produces staggered cuts around an adjacent pentanucleotide, resulting in 5'-terminal extensions. The nearer of the two cuts is five base-pairs to one side of the proposed recognition sequence. Because there are no common features within the cleaved sequences, the resultant pentanucleotide 5'-terminal extensions do not have the potential to anneal in a random manner as is the case with terminal extensions produced in sequences with 2-fold rotational symmetry.
from H. aegyptius and Providencia stuartii, Hae III and Pst I, respectively, were prepared by methods similar to those described by Roberts et al. (8) and were stored in 50% glycerol at -20°. Escherichia coli DNA polymerase I was purchased from the Boehringer Corp. Ltd. and T4 DNA polymerase was donated by R. Kamen and A. R. Coulson. DNA Sequencing. The procedure used was essentially that of Sanger and Coulson (9). The qX174 am3 RF DNA and complementary strand DNA were gifts of C. A. Hutchison III. Mapping of Hga I Sites in 4X174 DNA. Some Hga I cleavage sites were mapped by comparison of (a) the fragment pattern obtained by double digestion of OX 174 RF DNA with Hga I and a second, previously mapped restriction endonuclease (refs. 10 and 11; see Fig. 3) with (b) the fragment patterns obtained with either enzyme alone. Location of Cuts in Hga I Cleavage Sites. For a given Hga I cleavage site the cuts made in each DNA strand by Hga I were separately aligned with the plus and minus gel pattern used to define the DNA sequence (9) in the region of the cuts. The principle of the method for aligning the cuts with the DNA sequence, as applied to a hypothetical symmetrical restriction endonuclease site, is shown in Fig. 1. The method is equally applicable to nonsymmetrical cleavage sites, and is based on a method described previously (5). DNA polymerase is used to extend a primer (usually a restriction fragment) and copy the DNA template to a very limited degree in the presence of the four deoxyribonucleoside 5'-triphosphates, one of which is a-32P-labeled. The triphosphates and the polymerase are then removed. These steps are common to the site determination and the plus and minus procedures (9). Two aliquots are then taken and T4 DNA polymerase I used to greatly extend the primer in the presence of the four unlabeled deoxyribonucleoside 5' -triphosphates (the "cold chase" step). The T4 DNA polymerase in one of the two reactions is inactivated with phenol (I). Treatment of this product with the datum restriction endonuclease (i.e., that defining the end of the primer) and with the restriction endonuclease under investigation produces a radioactive (copy) fragment which, when subjected to gel elecAbbreviations: Alu I, Hae III, Hga I, Hap II, HindII, Hph I, Hha I, Mbo II, Pst I, Taq I refer to restriction endonucleases isolated from Arthrobacter luteus, Haemophilus aegyptius, H. gallinarum, H. aphrophilus, H. influenzae Rd, H. parahaemolyticus, H. haemolyticus, Moraxella bovis, Providencia stuartfl, and Thermnus aquatcus, respectively (3, 17). RF DNA is the replicative (duplex) form of kX174 DNA. * The term restriction endonuclease is applied to Hga I even though it has not been shown to be involved in biological restriction. t Present address: Department of Biochemistry, University of Bristol, Medical School, University Walk, Bristol BS8 1TD, England. * Present address: Department of Biochemistry, Faculty of Medicine, University of British Columbia, Vancouver, B.C., Canada V6T iW5.
MATERIALS AND METHODS Enzymes. Samples of Hga I were donated by R. J. Roberts and G. B. Petersen. Endonuclease from H. haemolytcus, Hha I, was a gift of R. J. Roberts. That from H. aphrophilus, Hap II, was purchased from Miles Laboratories Ltd. Endonucleases The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
3213
3214
Proc. Nati. Acad. Sci. USA 74 (1977)
Biochemistry: Brown and Smith
Restriction fragment 5' primer annealed 31 to template
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3'-G-T-A-T-A-C-5' cleaved symmetrically to give (i) flush ends, (ii) tetranucleotide 5'terminal extensions, and (iii) tetranucleotide 3'-terminal extensions. Details are given in Materials and Methods.
trophoresis alongside the plus and minus pattern from the same primer fragment, defines the site of cleavage in the copy strand of the DNA. The second site location reaction (II), containing the active T4 DNA polymerase, is also treated with the datum restriction endonuclease and the enzyme under investigation. Initially the same fragment of radioactive DNA is generated as in the first experiment. However, the T4 DNA polymerase then acts on it to convert it to the same length as the nonradioactive template strand fragment. If there is a 5'-terminal extension, then the radioactive copy strand is extended, by repair synthesis to the same length as the template fragment. If there is a flush end, then there is no change because the four unlabeled deoxyribonucleoside 5'-triphosphates present repair any 3' exonucleolytic degradation. In the case of a 3'-terminal extension, this is removed by the exonuclease activity of the T4 polymerase (12) until the ends are flush. Analysis of the product of this reaction by gel electrophoresis alongside the plus and minus pattern indirectly, but quite specifically, defines the cut in the second DNA strand. Experimental details for the application of this method to Hga I sites are given in Fig. 2. RESULTS Mapping of Some Hga I Cleavage Sites in 4X174 RF DNA. A determination of the complete 4X174 DNA fragment
map for Hga I was not attempted because of the complex pattern of products, which included a number of persistent partial digestion products (unpublished results). Instead, double digestion of OX174 RF DNA with Hga I and Pst I was carried out. Pst I, which cleaves OX174 DNA at a single precisely characterized site (5), was found to cleave two fragments in an Hga I partial digest pattern. These two fragments, approximately 400 and 195 nucleotide pairs long (the longer of which must be incompletely digested with Hga I), were cleaved to give fragments approximately 235 and 160 nucleotide pairs long. This indicates that there is an Hga I site 160 nucleotide pairs to one side of the Pst I site and Hga I sites 235 and 35 (195 minus 160) nucleotide pairs to the other side of the Pst I site. The correct orientation of these sites relative to the restriction fragment map of OX 174 DNA (Fig. 3) was determined during experiments to precisely locate the cuts at each Hga I cleavage site. Additional Hga I cleavage sites were mapped by double digestion of 4X174 RF DNA with Hga I and Hae III. Inter alia, Hae III fragments 5 and 8 (ref 11; Fig. 3) were cleaved in the double digest, indicating the presence of Hga I cleavage sites within these fragments. These sites were precisely mapped during cut-location experiments. Sequences Surrounding Six Hga I Cleavage Sites in 4X174 am3 RF DNA. The specific location of the cut in each strand of the DNA was established for each site by the method described in Materials and Methods and the legend to Fig. 2. Illustrative experiments, together with experimental details, are shown in Fig. 2 and five sequences, containing six sites, are summarized in Fig. 4. The four sequences with only one cleavage site all have staggered cuts resulting in fragments with pentanucleotide 5'-terminal extensions. The striking characteristic of these cleavage sites is that the pentanucleotide extensions do not contain any common sequences, nor do the regions immediately adjacent. However, the pentanucleotide sequence 5'-G-A-C-G-C-3'
3'-C-T-G-C-G-5' occurs five nucleotide pairs to the left of three of the cleavage sites and, inverted, occurs five nucleotide pairs to the right of the fourth cleavage site. In the fifth sequence this pentanucleotide sequence occurs twice, separated by two nucleotide pairs (Fig. 4). Staggered cuts are found, the nearer of each pair of cuts being five nucleotides to the left of the sequence 5'-G-C-G-T-C-3' 3'-C-G-C-A-G-5' An apparent additional cut is reproducibly found in one strand of this sequence; its origin is not understood. Examination of the sequence of OX174 viral strand DNA (11) reveals that two contiguous sequences 5'-G-A-C-G-C-3' occur 157 nucleotides to the left of the Pst I site, near where a Hga I site was mapped in double-digestion experiments. DISCUSSION This paper describes the characterization of Hga I cleavage sites by a method that is generally applicable. The method has been shown (unpublished results) to work with enzymes known to give fragments with flush ends (Hindll, Alu I), 3'-terminal extensions (Hae II)., or 5'-terminal extensions (Hap II) and with one enzyme that does not cleave within a symmetrical sequence (Hph I) (see ref. 3). The method requires no prior knowledge
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Proc. Natl. Acad. Sci. USA 74 (1977)
iA) stG -C -T -A
FIG. 2. Autoradiographs of plus and minus gel electrophoresis patterns for the determination of sequences cleaved by Hga I. Each plus and minus experiment was carried out as described by Sanger and Coulson (9), priming from a restriction enzyme cleavage site with complementary (nonviral) strand DNA as template. In addition, two aliquots (approximately 0.1 pmol of DNA) of the 32P-labeled random-length product from the first stage of the plus and minus reaction (9) were incubated for 30 min at 370 with T4 DNA polymerase (approximately 0.02 unit) and all four unlabeled deoxyribonucleoside 5'-triphosphates (0.4 mM) in buffer B (50 mM NaCl/6.6 mM MgC12/6.6 mM 2-mercaptoethanol/6.6 mM Tris.HCl, pH 7.4). One of the aliquots (I) was diluted with water (10 Ml) and treated with water-saturated phenol (25 gl). The aqueous layer was washed with ether (five times with 250 Ml) and the residual ether was removed by blowing air over the sample. The buffer concentration was adjusted to the concentration of buffer B. Both aliquots were then treated with Hga I and the same nuclease that was used to generate the plus and minus pattern. The two aliquots, and an aliquot of the product from the first stage of the plus and minus reaction (channel 0), were denatured and analyzed alongside the plus and minus experiment by electrophoresis through a 12% acrylamide gel containing 7 M urea/90 mM Tris-borate, pH 8.3/2.5 mM EDTA. The gel was then autoradiographed. The aliquot treated with phenol (channelI) defines the site of cleavage in the radioactive (copy) strand. The other aliquot (channel II) defines the cleavage site in the template strand. The autoradiographs shown were obtained after 5 days of exposure in order
is within a region of DNA whose nucleotide sequence can be determined (i.e., is within 100 nucleotides of a different restriction endonuclease cleavage site that can be used for primed synthesis). The method described here is modified from the version described in preliminary form (5) in that a "cold chase" step is used in the preparation of aliquot I (Fig. 1). This is to ensure that the DNA in aliquot I is a substrate for restriction enzymes such as Alu I and Hph I, which are not active on duplex DNA in the presence of extensive regions of single-stranded DNA (ref. 13, and unpublished results). In the experiments described here the cleavage sites are aligned with the plus and minus gel patterns obtained by the method of Sanger and Coulson (9). However, equivalent gel electrophoresis methods for DNA sequence determination (14) can be used for the sequence alignment.
Hga I resembles class II enzymes in its cofactor requirements (1,3), but like Hph 1 (6) and Mbo II (7) it cuts to one side of an asymmetrical pentanucleotide recognition sequence. The presumed recognition sequence is 5'-G-A-C-G-C-3' 3'-C-T-G-C-G-5' and the cleavage site is staggered, giving rise to pentanucleotide 5'-terminal extensions. The cut in the DNA strand containing the sequence 5'-G-A-C-G-C-3' is five nucleotides to the 3' (right) side of this sequence. The cut in the complementary strand is 10 nucleotides to the 5' (right) side of the sequence 3'-C-T-G-C-G-5'. Examination of a model of the DNA double helix shows that the cut sites lie on the same side of the helix, and the enzyme could function by aligning itself with the recognition site on the same side of the helix. Examination of the sequence of OX X174 am3 viral DNA (11) reveals that the sequences G-A-C-G-C and G-C-G-T-C occur 14 times. This is in agreement with the number of fragments produced on digestion of (X174 RF DNA with Hga I (4). However, a complete Hga I cleavage map has not been constructed due to the presence of persistent nonstoichiometric digestion products (ref. 4 and unpublished results). There may be several possible sources of such products. First, there are two sets of closely spaced recognition sequences, the pair separated by two nucleotides shown in Figs. 2 and 4, and a pair of contiguous recognition sites 157 nucleotides to the left of the Pst I site (nucleotides 5209-5219; ref. 11). Such closely spaced sites result in stable nonstoichiometric digestion products, as has been shown with Mbo 11 (7). In addition, there may be differences in the rates of cleavage of individual restriction sites (15), and this may be very marked for enzymes that do not cleave at their recognition sites. This has been seen in the digestion of OX174 RF DNA with Hph I (C. A. Hutchison III and M. Smith, unpublished results), and results in persistent partial digestion to show all the bands in the sequence. The bands in channels I and II, due to cleavage with Hga I, are therefore very intense. The correct alignment of these bands with the plus and minus sequence was obtained in 16- to 24-hr autoradiographs of the same gel. (A) Fragment 5 primer of the endonuclease from Arthrobacter luteus, Alu I, complementary strand template, cleaved at the Hha I 8b/4 site (Fig. 3). The detailed sequence is shown in Fig. 4 (ii). (B) fragment 3 primer of the endonuclease from Thermus aquaticus, Taq I, complementary strand template, cleaved at the Hap II 3/2 site (Fig. 3). The detailed sequence is shown in Fig. 4 (iv). (C) Hae III fragment 7 primer, complementary strand template, cleaved at the Hae III 7/5 site (Fig. 3). The detailed sequence is shown in Fig. 4 (v).
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Proc.. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Brown and Smith 0
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