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DNAase I footprinting of restriction enzymes
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 779-785
Vol. 155, No. 2, 1988 September 15, 1988
DNAase
I Footprinting
of R e s t r i c t i o n
Enzymes
K e i t h R. Fox Dept.
of P h y s i o l o g y and P h a r m a c o l o g y U n i v e r s i t y of S o u t h a m p t o n Bassett Crescent East S o u t h a m p t o n $09 3TU U.K.
Received July 18, 1988 DNAase I footprintinq of restriction enzymes has been achieved by using calcium containing digestion buffers so that the enzymes bind to but do not cleave DNA. EccRI produces a footprint 17 bases long, overestimating the region of contact with DNA by about 7-8 base pairs. Restriction enz-/mes HaeIII and HirPl generate smaller footprints of 15 and 13 base pairs respectively. © 1988AcademicP..... Inc.
Type II restriction enzymes are endonucleases which recognize and cleave specific sequences in double-stranded DNA which are typically four to eight base pairs long. They are widely used as tools in molecular biology. Although a wide number of such enzymes have been isolated and many are commercially available,
little is known about the details of
their interaction with DNA. One notable exception, about which a great deal is known is the enzyme EcoRI for which a crystal structure has been determined bound to an oligonucleotide containing its recognition sequence G ~ T r C
[1,2]. In this structure the bulk of the protein molecule
is located in the DNA major groove and sequence selectivity is achieved by 12 hydrogen b~nds to the base pairs. Although the DNA fragment (12 base pairs) was longer than the DNA binding face of the enzyme, it matched the net width of 'the protein dimer so that there appeared to be a solvent gap between the ends of the dodecanucleotide and the protein. Previous studies had shown that, although base recognition is restricted to the hexanucleotide GAAI~fC, such a short fragment is not a good substrate for the epxyme; the Km for cleaving an octamer is about I00 times greater than for a decamer or longer DNA substrate [3]. Footprinting studies with this enzyme using the purine-specific reaction with dimethylsulphate have shown that the enzyme only protects those purines which form part of the recognition sequence [4]. In contrast the enzyme protects two phosphate residues 5' external to this sequence from ethylation. It therefore appears that some protein-DNA interactions occur outside the canonical recognit ion sequence.
779
0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EcoRl, in common with many other restriction enzymes, requires magnesium for activity; this can not be replaced with calcium, The most commonly used footprinting probe is DNAase I. This enzyme also requires divalent metal ions, though calcium can replace magnesium. By using calcium containing buffers it is therefore possible to control conditions so that the footprinting probe cuts DNA while the restriction enzyme can only bind. The structure of DNAase I has been determined [5,6] and it is known to bind to DNA via the minor groove. The purpose of this study is to describe a simple method for footprinting restriction enzymes which can be applied to very small quantities of material which need not necessarily be pure, such as commercial preparations. In order to calibrate the results with a known structure the results for EccRl are described first.
MATERIALS and METHODS Restriction erlzymes were purchased from either New Ehgland Biolabs, Amersham, Northumbria Bioloqicals or Sigma. Deoxyribonuclease I (DNAase I) was purchased from Sigma and stored as a 7200 units/ml stock golution in O.15M NaCI containing imM MqCI2. It was stored at -20°C and diluted to working concentrations immediately before use. An end labelled 102-base-pair DNA fragment containing the EccRl recognition site was isolated from plasmid pBR322 by cutting with HiralIII, labelling the 3' ends with I~-32p]dATP using reverse transcriptase, or the 5'-ends with [Y-3ZP]ATP using polynucleotide kinase. The labelled DNA was then cut with AatII and the 102 base pair fragment separated on a 6% polyacrylamide qel. The tyzT DNA fragment was isolated and labelled at the 3' end of the EccRl site (hot) or the AvaI site (top) as previously descriDed [7]. All DNA fragments were disolved in lOmM Tris-HCl pH 8.0 containing O. ImM IK)TA. Although DNAase I may not be the most exact probe for assessing the binding site size of proteins on DNA, on account of its bulk, it is widely used as a footprinting tool, and its mode of binding to DNA has been well documented [5,6]. Better probes might be hydroxyl radicals [8] or MPE-Fe 2~ [9], which generate even ladders of bards and precisely map out protein-DNA contacts. However,the former can not be applied to commercial enzyme preparations since these contain glycerol as a stabilizer which inhibits the free radical reaction. MPE-Fe 2÷ acts by intercalating its methidium chromophore between the base pairs and so induces DNA structural changes. It is also less sensitive to weakly hound sites. DNAase I footprinting was performed by mixing 3~i DNA (about lOpmol in base pairs) with 3~I of restriction enzyme as suppled. To this was added 2~I of DNAase I at an appropriate concentration dissolved in 5raM CaCI2, 20ram NaCl. Samples were removed from the the diqestion after 1,5 and 30 rains and the reaction stopped by adding 3ul 80% formamide containing 10ram ]~)TA. Samples were heated at 100°C prior to electrophoresis on 8% polyacrylamide gels containing 8M urea. Gels were transferred to Whatman 3MM paper, dried under vacuum and subjected to autoradiography at -70~C with an intensifying screen. Bands in the autoradiographs were assigned by comparison with Maxam-Gilbert G-specific sequencing lanes. The reaction with piperidine used to generate these marker lanes leaves DNA fragments with phosphate groups at both the 3' and 5' side of the cutting site. In contrast DNAase I, which cuts the 03'-phosphate bond, leaves a 3'-hydroxyl and a 5' phosphate group. As a result corresponding fragments labelled at the 5'-end run slower in the DNAase I treated lanes than the marker lanes, this difference in mobility is more pronounced for shorter fragments.
780
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PZSULTS and DISCUSSION DNAase I digestions of the 102-base-pair DNA fragment labelled at either end in the presence and absence or EcoRl are presented in Figtn'e I. It can be seen that in the presence of calcium the restriction e,~/me is unable to cleave the DNA and that a single blockage site is visible on each DNA strand from position 4353 through to position 8 on the 5'-end labelled strand ard from position 4356 through to position I0 on the
3'-end l.abelled strand. The protected region is staggered across the
5 ' - e n d labelled
3 ' - e n d labelled b
CON
EcoR1
o
--4330 --4340
++,++++
4360-I --
~++ ++++--I
~0--
Fiqure 1 DNAase I footprinting of EcoRl on the 102-base-pair DNA fragment labelled at either the 3' or 5' end. For the 5'-labelled DNA the tracks labelled 1,5 and 30 correspond to the length of enzyme digestion in minutes. 'the 3'-labelled tracks were each digested for 5 rain. Tracks labelled 'G' represent dimethylsulphate-piperidine markers specific for guanine. The vertical numbers refer to the numbering scheme for pI~322 [15] and correspond to the bond cut by DNAase I (which does not correspond exactly to the C-track for the 5'-end labelled fragment).
781
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
strands of the DNA double helix by 2-3 bonds in the 3' direction as expected for an enzyme which cuts from the minor groove. The DNA protected from DNAase I attack by EcoRl is therefore 17-18 bases long. This compares with a recognition sequence of six base pairs and DNA-contact regions of ten bases seen in the crystal structure.
The larger value
determined by footprinting is due to the bulk of DNAase I since this enzyme covers at least four base-pairs to the 5' side and 6 base-pairs to the 3 ' side of its cutting s~te on one side of the DNA double helix. As previously noted, if a phosphodiester bond is accessible to DNAase I cleavage then this implies that the protein being footprinted is not in the minor groove following the 3' direction or the major groove in the 5' direction [5]. It appears then that these factors result in DNAase I overestimating the size of a major groove binding protein by 7-8 base pairs, This is one of the first examples of a comparison between DNAase I footprinting and a crystal structure of a protein bottnd to a DNA fragment. It is worth considering what effect a minor-groove binding protein of similar size would have on DNAase I cleavage. Opposing phosphates across the DNA major groove are separated by about 7 base pairs, whereas 3 base pairs separate phosphates across the minor groove. A major groove binding protein will affect phosphates on the adjacent minor groove which are only 3 bases away whereas a minor groove binding protein may have contacts with opposing phosphates which are 7 bases removed from its binding site. It may therefore be anticipated that the footprinting pattern generated by a protein forming specific contacts in the minor groove would be greater by an additional 8 bases pairs. One bond on each DNA strand shows an enhanced rate of DNAase I cleavage in the presence of EcoRl, visible at position 12 on the 3' labelled strand and position 4350 on the 5' labelled strand. These enhancements are 14 bases away from the restriction enzyme cutting sites on each DNA strand. Since these enhancement are single stranded events they are unlikely to be due to any distortions in DNA structure affecting the minor groove width, and are probably the result of changes in the precise orientation of this phosphate group, facing the enzyme across the DNA minor groove. Figure 2 shows DNAase I footprinting patterns on tyrT DNA in the presence of the restriction enzymes HirPl (G/CGC) and HaeIII (GG/CC). In both instances cutting by the restriction enzymes has been largely eliminated by working in the calcium containing buffer, while the enzymes ability to bind to DNA has not been seriously affected, as evidenced by the protection patterns produced arour~ their recognition sites. HaeIII has one cutting site on the DNA fragment located at position 121 and protects 15 bases between 115-129 from DNAase I cleavage.
782
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS CON HinP1 Haelll 15301G11 53011530
Figure 2 DNAase I digestion of tyrT DNA labelled at the 3'-end of the avaI site in the presence of HirPl and HaeIII. Time in minutes after the addition of enzyme is shown at the top of each gel lane. The track labelled 'G' is a dimethylsulphate- piperidine marker specific for guanine, Vertical numbers refer to the numbering scheme for tyrT DNA [7].
HirPl has two recognition sites on this fragment and cuts at positions 94 and 75, each of these generates a footprint 13 bases long visible between posit~ons 90-102 and 71-83. The results obtained with other restriction enzymes using this footprinting technique
are presented in
Table i. In certain instances the restriction enzyme cutting was not completely inhibited by omitting magnesium (see for example the results for HaeIII in Figure 2). This could be because either the enzyme preparation contains sufficient magnesium to allow a slow rate of cleavage ,or that it retains very low activity in the presence of calcium. Footprinting experiments with Bgpl and /~aI were unsuccessul, no pattern of protection was obtained. This could be merely because the absolute concentration of enzyme in these commercial preparations was too low.
783
Vol. 155, No. 2, 1988
]~%zyme
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DNA fragment
cutting site region protected
number bases protected
EcoRl 102-mer (3') (G/AATrC) 102-mer (5')
4360
4356-i0
17
2
4353-49
18
HaeIII (GG/CC)
102-mer (3')
4344
4337-4351
15
tyrT (top)
121
115-129
14
tyrT (top)
94
90-102
13
tyrT (top)
75
71-83
13
t ~ T (~t)
~
~
16
t~T
(~t)
94
88-i~
17
tyrT (top)
77
68-88
21
HinPl (G/CGC)
C~I C~/~)
FnuDI I (0S/OS)
Commercial specific activities are variable so that the absolute enzyme concentration could vary over a wide range. It must therefore be emphasized that a failure to obtain a footprinting pattern can not be
rigorously interpreted. The uncertainty in the absolute enzyme concentration also means that the results can only be qualitative
(as are
most footprinting experiments) and that quantitative conclusions can not be inferred. From this data it can be seen that H/nPI and HaeIII produce smaller footprints than Ecd~l. If we assume that each of these enzymes interact with DNA in a similar fashion and note that DNAase I overestimates the EccRI site size by 7-8 base pairs, then it appears that HaeIII possesses a D N A binding region 3 bases less than EcdRl while HirPl is 4.5 base shorter. From this calculation it seems that the actual DNA binding site size for HirPl (5 base pairs) may be little bigger than the four base pair recognition sequence. One might therefore guess that cutting by this enzyme will not be affected by the nature of the flanking sequences, with which it has little or no physical interaction. This is not the case; the relative probablities for this enzyme cutting several sites on a long DNA fragment are nearly identical to those of ]/~aI, (unpublished observations) which displays a preference for cutting at sequences of the type ~
[I0]. This would suggest that variations
cutting rate at sequences surrounded by different flanking regions are not determined by the binding reaction itself, but by the catalytic activity
784
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the enzyme, presumably as the
result of the ability to distort the
local DNA structure to a cleavable conformation. Indeed this is in agreement with the explanation for the differences in cutting by EccRl [II]. The preliminary results with FnuDII suggest that this enzyme is much larger, or interacts with the DNA in a different fashion, possibly via the minor groove. It has been suggested that HaeIII has specific contacts with base substituents in both major and minor grooves [12], while little is known about the other enzymes used in this study. In conclusion it has been shown that restriction enzyme binding site sizes on DNA can not be predicted from the recognition sequences themselves, qTnjs has important consequences for interpretting experiments in which restriction enzymes are used as probes for drug binding site on DNA [13,14]. Clearly HirP! will be able to approach more closely to a bound ligand than FnuDII.
A£~2~O~ENEN2~ This work was supported by grants form the Wellcome Trust and the Medical Research Council.
P217ERENCES [i]. Frederick, C.A., Grable, J., Melia, M., Samudzi, C., Jen-Jacobsen, L., Wang, B.-C., Greene, P., Boyer, H.W. and Rosenberg, J.M. (1984). Nature 309, 327-331. [2]. McClarin, J.A., Frederick, C.A.,Wang, B.-C., Greene, P., Boyer, H.W., Grable, J° and Rosenberg, J.M. (1986). Science 234, 1526-1541. [3] ."Green, P.J., Poonian, M.S., Nussbaun, A.L., Tobias, L., Garfin, D.E., Boyer, H.W., & Goodmen, H.M. (1975). J. Mol. Biol. 99, 237-261. [4]. Lu, A.-L., Jack, W.E. & Modrich, P. (1981) J. Biol. Chem. 256, 13200-13206, [5]. Suck, D. & Oefner, C. (1986) Nature 321, 620-625. [6]. Suck, D., Lahm, A. & Oefner, C. (1988) Nature, 332, 464-468. [7]. Drew, H.R. & Travers, A.A (1984) Cell 37, 491-502. [8]. Tullius, T.D. & Dombroski, B.A (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5469-5473 [9]. Van l>/ke, M.W. & Dervan, P.B. (1983) Nucl. Acids Res. ii, 5555-5567. [I0]. Drew, H.R. & Travers, A.A. (1985) Nucl. Acids Res. 13, 4445-4467. [ii]. Halford, S.E. & Johnson, N.R. (1980) Biochem. J. 191, 593-603. [12]. Wolfes, H. Fleiss, A. & Pingoud, A. (1985) Eur. J. Biochem. 150, 105-110. [13]. Nosikov, V.V., ]3raga, E.A., Karlishev, A.V. Zhuze, A. & Polyanovsky, O.L. (1976) Nucl. Acids Res. 4, 367-371. [14]. Malcolm, A.D.B. Moffatt, J.R., Fox, K.R. & Waring, M.J. (1982) Biochim. Biophys. Acta. 699, 211-216. [15]. Sutcliffe, J.G. (1979) Cold Spring Harbour, Symp. Quant. Biol. 43, 77-90.
785
Vol. 155, No. 2, 1988 September 15, 1988
DNAase
I Footprinting
of R e s t r i c t i o n
Enzymes
K e i t h R. Fox Dept.
of P h y s i o l o g y and P h a r m a c o l o g y U n i v e r s i t y of S o u t h a m p t o n Bassett Crescent East S o u t h a m p t o n $09 3TU U.K.
Received July 18, 1988 DNAase I footprintinq of restriction enzymes has been achieved by using calcium containing digestion buffers so that the enzymes bind to but do not cleave DNA. EccRI produces a footprint 17 bases long, overestimating the region of contact with DNA by about 7-8 base pairs. Restriction enz-/mes HaeIII and HirPl generate smaller footprints of 15 and 13 base pairs respectively. © 1988AcademicP..... Inc.
Type II restriction enzymes are endonucleases which recognize and cleave specific sequences in double-stranded DNA which are typically four to eight base pairs long. They are widely used as tools in molecular biology. Although a wide number of such enzymes have been isolated and many are commercially available,
little is known about the details of
their interaction with DNA. One notable exception, about which a great deal is known is the enzyme EcoRI for which a crystal structure has been determined bound to an oligonucleotide containing its recognition sequence G ~ T r C
[1,2]. In this structure the bulk of the protein molecule
is located in the DNA major groove and sequence selectivity is achieved by 12 hydrogen b~nds to the base pairs. Although the DNA fragment (12 base pairs) was longer than the DNA binding face of the enzyme, it matched the net width of 'the protein dimer so that there appeared to be a solvent gap between the ends of the dodecanucleotide and the protein. Previous studies had shown that, although base recognition is restricted to the hexanucleotide GAAI~fC, such a short fragment is not a good substrate for the epxyme; the Km for cleaving an octamer is about I00 times greater than for a decamer or longer DNA substrate [3]. Footprinting studies with this enzyme using the purine-specific reaction with dimethylsulphate have shown that the enzyme only protects those purines which form part of the recognition sequence [4]. In contrast the enzyme protects two phosphate residues 5' external to this sequence from ethylation. It therefore appears that some protein-DNA interactions occur outside the canonical recognit ion sequence.
779
0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EcoRl, in common with many other restriction enzymes, requires magnesium for activity; this can not be replaced with calcium, The most commonly used footprinting probe is DNAase I. This enzyme also requires divalent metal ions, though calcium can replace magnesium. By using calcium containing buffers it is therefore possible to control conditions so that the footprinting probe cuts DNA while the restriction enzyme can only bind. The structure of DNAase I has been determined [5,6] and it is known to bind to DNA via the minor groove. The purpose of this study is to describe a simple method for footprinting restriction enzymes which can be applied to very small quantities of material which need not necessarily be pure, such as commercial preparations. In order to calibrate the results with a known structure the results for EccRl are described first.
MATERIALS and METHODS Restriction erlzymes were purchased from either New Ehgland Biolabs, Amersham, Northumbria Bioloqicals or Sigma. Deoxyribonuclease I (DNAase I) was purchased from Sigma and stored as a 7200 units/ml stock golution in O.15M NaCI containing imM MqCI2. It was stored at -20°C and diluted to working concentrations immediately before use. An end labelled 102-base-pair DNA fragment containing the EccRl recognition site was isolated from plasmid pBR322 by cutting with HiralIII, labelling the 3' ends with I~-32p]dATP using reverse transcriptase, or the 5'-ends with [Y-3ZP]ATP using polynucleotide kinase. The labelled DNA was then cut with AatII and the 102 base pair fragment separated on a 6% polyacrylamide qel. The tyzT DNA fragment was isolated and labelled at the 3' end of the EccRl site (hot) or the AvaI site (top) as previously descriDed [7]. All DNA fragments were disolved in lOmM Tris-HCl pH 8.0 containing O. ImM IK)TA. Although DNAase I may not be the most exact probe for assessing the binding site size of proteins on DNA, on account of its bulk, it is widely used as a footprinting tool, and its mode of binding to DNA has been well documented [5,6]. Better probes might be hydroxyl radicals [8] or MPE-Fe 2~ [9], which generate even ladders of bards and precisely map out protein-DNA contacts. However,the former can not be applied to commercial enzyme preparations since these contain glycerol as a stabilizer which inhibits the free radical reaction. MPE-Fe 2÷ acts by intercalating its methidium chromophore between the base pairs and so induces DNA structural changes. It is also less sensitive to weakly hound sites. DNAase I footprinting was performed by mixing 3~i DNA (about lOpmol in base pairs) with 3~I of restriction enzyme as suppled. To this was added 2~I of DNAase I at an appropriate concentration dissolved in 5raM CaCI2, 20ram NaCl. Samples were removed from the the diqestion after 1,5 and 30 rains and the reaction stopped by adding 3ul 80% formamide containing 10ram ]~)TA. Samples were heated at 100°C prior to electrophoresis on 8% polyacrylamide gels containing 8M urea. Gels were transferred to Whatman 3MM paper, dried under vacuum and subjected to autoradiography at -70~C with an intensifying screen. Bands in the autoradiographs were assigned by comparison with Maxam-Gilbert G-specific sequencing lanes. The reaction with piperidine used to generate these marker lanes leaves DNA fragments with phosphate groups at both the 3' and 5' side of the cutting site. In contrast DNAase I, which cuts the 03'-phosphate bond, leaves a 3'-hydroxyl and a 5' phosphate group. As a result corresponding fragments labelled at the 5'-end run slower in the DNAase I treated lanes than the marker lanes, this difference in mobility is more pronounced for shorter fragments.
780
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PZSULTS and DISCUSSION DNAase I digestions of the 102-base-pair DNA fragment labelled at either end in the presence and absence or EcoRl are presented in Figtn'e I. It can be seen that in the presence of calcium the restriction e,~/me is unable to cleave the DNA and that a single blockage site is visible on each DNA strand from position 4353 through to position 8 on the 5'-end labelled strand ard from position 4356 through to position I0 on the
3'-end l.abelled strand. The protected region is staggered across the
5 ' - e n d labelled
3 ' - e n d labelled b
CON
EcoR1
o
--4330 --4340
++,++++
4360-I --
~++ ++++--I
~0--
Fiqure 1 DNAase I footprinting of EcoRl on the 102-base-pair DNA fragment labelled at either the 3' or 5' end. For the 5'-labelled DNA the tracks labelled 1,5 and 30 correspond to the length of enzyme digestion in minutes. 'the 3'-labelled tracks were each digested for 5 rain. Tracks labelled 'G' represent dimethylsulphate-piperidine markers specific for guanine. The vertical numbers refer to the numbering scheme for pI~322 [15] and correspond to the bond cut by DNAase I (which does not correspond exactly to the C-track for the 5'-end labelled fragment).
781
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
strands of the DNA double helix by 2-3 bonds in the 3' direction as expected for an enzyme which cuts from the minor groove. The DNA protected from DNAase I attack by EcoRl is therefore 17-18 bases long. This compares with a recognition sequence of six base pairs and DNA-contact regions of ten bases seen in the crystal structure.
The larger value
determined by footprinting is due to the bulk of DNAase I since this enzyme covers at least four base-pairs to the 5' side and 6 base-pairs to the 3 ' side of its cutting s~te on one side of the DNA double helix. As previously noted, if a phosphodiester bond is accessible to DNAase I cleavage then this implies that the protein being footprinted is not in the minor groove following the 3' direction or the major groove in the 5' direction [5]. It appears then that these factors result in DNAase I overestimating the size of a major groove binding protein by 7-8 base pairs, This is one of the first examples of a comparison between DNAase I footprinting and a crystal structure of a protein bottnd to a DNA fragment. It is worth considering what effect a minor-groove binding protein of similar size would have on DNAase I cleavage. Opposing phosphates across the DNA major groove are separated by about 7 base pairs, whereas 3 base pairs separate phosphates across the minor groove. A major groove binding protein will affect phosphates on the adjacent minor groove which are only 3 bases away whereas a minor groove binding protein may have contacts with opposing phosphates which are 7 bases removed from its binding site. It may therefore be anticipated that the footprinting pattern generated by a protein forming specific contacts in the minor groove would be greater by an additional 8 bases pairs. One bond on each DNA strand shows an enhanced rate of DNAase I cleavage in the presence of EcoRl, visible at position 12 on the 3' labelled strand and position 4350 on the 5' labelled strand. These enhancements are 14 bases away from the restriction enzyme cutting sites on each DNA strand. Since these enhancement are single stranded events they are unlikely to be due to any distortions in DNA structure affecting the minor groove width, and are probably the result of changes in the precise orientation of this phosphate group, facing the enzyme across the DNA minor groove. Figure 2 shows DNAase I footprinting patterns on tyrT DNA in the presence of the restriction enzymes HirPl (G/CGC) and HaeIII (GG/CC). In both instances cutting by the restriction enzymes has been largely eliminated by working in the calcium containing buffer, while the enzymes ability to bind to DNA has not been seriously affected, as evidenced by the protection patterns produced arour~ their recognition sites. HaeIII has one cutting site on the DNA fragment located at position 121 and protects 15 bases between 115-129 from DNAase I cleavage.
782
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS CON HinP1 Haelll 15301G11 53011530
Figure 2 DNAase I digestion of tyrT DNA labelled at the 3'-end of the avaI site in the presence of HirPl and HaeIII. Time in minutes after the addition of enzyme is shown at the top of each gel lane. The track labelled 'G' is a dimethylsulphate- piperidine marker specific for guanine, Vertical numbers refer to the numbering scheme for tyrT DNA [7].
HirPl has two recognition sites on this fragment and cuts at positions 94 and 75, each of these generates a footprint 13 bases long visible between posit~ons 90-102 and 71-83. The results obtained with other restriction enzymes using this footprinting technique
are presented in
Table i. In certain instances the restriction enzyme cutting was not completely inhibited by omitting magnesium (see for example the results for HaeIII in Figure 2). This could be because either the enzyme preparation contains sufficient magnesium to allow a slow rate of cleavage ,or that it retains very low activity in the presence of calcium. Footprinting experiments with Bgpl and /~aI were unsuccessul, no pattern of protection was obtained. This could be merely because the absolute concentration of enzyme in these commercial preparations was too low.
783
Vol. 155, No. 2, 1988
]~%zyme
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DNA fragment
cutting site region protected
number bases protected
EcoRl 102-mer (3') (G/AATrC) 102-mer (5')
4360
4356-i0
17
2
4353-49
18
HaeIII (GG/CC)
102-mer (3')
4344
4337-4351
15
tyrT (top)
121
115-129
14
tyrT (top)
94
90-102
13
tyrT (top)
75
71-83
13
t ~ T (~t)
~
~
16
t~T
(~t)
94
88-i~
17
tyrT (top)
77
68-88
21
HinPl (G/CGC)
C~I C~/~)
FnuDI I (0S/OS)
Commercial specific activities are variable so that the absolute enzyme concentration could vary over a wide range. It must therefore be emphasized that a failure to obtain a footprinting pattern can not be
rigorously interpreted. The uncertainty in the absolute enzyme concentration also means that the results can only be qualitative
(as are
most footprinting experiments) and that quantitative conclusions can not be inferred. From this data it can be seen that H/nPI and HaeIII produce smaller footprints than Ecd~l. If we assume that each of these enzymes interact with DNA in a similar fashion and note that DNAase I overestimates the EccRI site size by 7-8 base pairs, then it appears that HaeIII possesses a D N A binding region 3 bases less than EcdRl while HirPl is 4.5 base shorter. From this calculation it seems that the actual DNA binding site size for HirPl (5 base pairs) may be little bigger than the four base pair recognition sequence. One might therefore guess that cutting by this enzyme will not be affected by the nature of the flanking sequences, with which it has little or no physical interaction. This is not the case; the relative probablities for this enzyme cutting several sites on a long DNA fragment are nearly identical to those of ]/~aI, (unpublished observations) which displays a preference for cutting at sequences of the type ~
[I0]. This would suggest that variations
cutting rate at sequences surrounded by different flanking regions are not determined by the binding reaction itself, but by the catalytic activity
784
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the enzyme, presumably as the
result of the ability to distort the
local DNA structure to a cleavable conformation. Indeed this is in agreement with the explanation for the differences in cutting by EccRl [II]. The preliminary results with FnuDII suggest that this enzyme is much larger, or interacts with the DNA in a different fashion, possibly via the minor groove. It has been suggested that HaeIII has specific contacts with base substituents in both major and minor grooves [12], while little is known about the other enzymes used in this study. In conclusion it has been shown that restriction enzyme binding site sizes on DNA can not be predicted from the recognition sequences themselves, qTnjs has important consequences for interpretting experiments in which restriction enzymes are used as probes for drug binding site on DNA [13,14]. Clearly HirP! will be able to approach more closely to a bound ligand than FnuDII.
A£~2~O~ENEN2~ This work was supported by grants form the Wellcome Trust and the Medical Research Council.
P217ERENCES [i]. Frederick, C.A., Grable, J., Melia, M., Samudzi, C., Jen-Jacobsen, L., Wang, B.-C., Greene, P., Boyer, H.W. and Rosenberg, J.M. (1984). Nature 309, 327-331. [2]. McClarin, J.A., Frederick, C.A.,Wang, B.-C., Greene, P., Boyer, H.W., Grable, J° and Rosenberg, J.M. (1986). Science 234, 1526-1541. [3] ."Green, P.J., Poonian, M.S., Nussbaun, A.L., Tobias, L., Garfin, D.E., Boyer, H.W., & Goodmen, H.M. (1975). J. Mol. Biol. 99, 237-261. [4]. Lu, A.-L., Jack, W.E. & Modrich, P. (1981) J. Biol. Chem. 256, 13200-13206, [5]. Suck, D. & Oefner, C. (1986) Nature 321, 620-625. [6]. Suck, D., Lahm, A. & Oefner, C. (1988) Nature, 332, 464-468. [7]. Drew, H.R. & Travers, A.A (1984) Cell 37, 491-502. [8]. Tullius, T.D. & Dombroski, B.A (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5469-5473 [9]. Van l>/ke, M.W. & Dervan, P.B. (1983) Nucl. Acids Res. ii, 5555-5567. [I0]. Drew, H.R. & Travers, A.A. (1985) Nucl. Acids Res. 13, 4445-4467. [ii]. Halford, S.E. & Johnson, N.R. (1980) Biochem. J. 191, 593-603. [12]. Wolfes, H. Fleiss, A. & Pingoud, A. (1985) Eur. J. Biochem. 150, 105-110. [13]. Nosikov, V.V., ]3raga, E.A., Karlishev, A.V. Zhuze, A. & Polyanovsky, O.L. (1976) Nucl. Acids Res. 4, 367-371. [14]. Malcolm, A.D.B. Moffatt, J.R., Fox, K.R. & Waring, M.J. (1982) Biochim. Biophys. Acta. 699, 211-216. [15]. Sutcliffe, J.G. (1979) Cold Spring Harbour, Symp. Quant. Biol. 43, 77-90.
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