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The monomeric homing endonuclease PI-SceI has two catalytic centres for cleavage of the two strands of its DNA substrate

1999; Springer Nature; Volume: 18; Issue: 24 Linguagem: Inglês

10.1093/emboj/18.24.6908

1460-2075

Frauke Christ,

Bacterial Genetics and Biotechnology

Article15 December 1999free access The monomeric homing endonuclease PI-SceI has two catalytic centres for cleavage of the two strands of its DNA substrate Frauke Christ Frauke Christ Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany Search for more papers by this author Sylvia Schoettler Sylvia Schoettler Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany Search for more papers by this author Wolfgang Wende Wolfgang Wende Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany Search for more papers by this author Shawn Steuer Shawn Steuer Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany Search for more papers by this author Alfred Pingoud Alfred Pingoud Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany Search for more papers by this author Vera Pingoud Corresponding Author Vera Pingoud Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany Search for more papers by this author Frauke Christ Frauke Christ Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany Search for more papers by this author Sylvia Schoettler Sylvia Schoettler Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany Search for more papers by this author Wolfgang Wende Wolfgang Wende Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany Search for more papers by this author Shawn Steuer Shawn Steuer Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany Search for more papers by this author Alfred Pingoud Alfred Pingoud Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany Search for more papers by this author Vera Pingoud Corresponding Author Vera Pingoud Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany Search for more papers by this author Author Information Frauke Christ1, Sylvia Schoettler1, Wolfgang Wende1, Shawn Steuer1, Alfred Pingoud1 and Vera Pingoud 1 1Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:6908-6916https://doi.org/10.1093/emboj/18.24.6908 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The monomeric homing endonuclease PI-SceI cleaves the two strands of its DNA substrate in a concerted manner, which raises the question of whether this enzyme harbours one or two catalytic centres. If PI-SceI has only one catalytic centre, one would expect that cross-linking enzyme and substrate should prevent reorientation of the enzyme required to perform the second cut after having made the first cut: PI-SceI, however, when cross-linked to its substrate, is able to cleave both DNA strands. If PI-SceI has two catalytic centres, one would expect that it should be possible to inactivate one catalytic centre by mutation and obtain a variant with preference for a substrate nicked in one strand; such variants have been found. The structural homology between the catalytic domain of PI-SceI having a pseudo 2-fold symmetry, and I-CreI, a homodimeric homing endonuclease, suggests that in PI-SceI active site I, which attacks the top strand, comprises Asp218, Asp229 and Lys403, while Asp326, Thr341 and Lys301 make up active site II, which cleaves the bottom strand. Cleavage experiments with modified oligodeoxynucleotides and metal ion mapping experiments demonstrate that PI-SceI interacts differently with the two strands at the cleavage position, supporting a model of two catalytic centres. Introduction Homing endonucleases are enzymes which catalyse a highly specific double strand cleavage within an extended recognition sequence and thereby induce a double strand break repair that leads to insertion of their gene into an allele which lacks it (Lambowitz and Belfort, 1993; Mueller et al., 1993). Homing endonucleases are encoded in introns or as protein introns (inteins) of prokaryotes, eukaryotes and archaea (reviewed in Dujon, 1989; Belfort and Roberts, 1997). The intein-encoded homing endonucleases combine two catalytic functions in one molecule, endonucleolytic activity and protein splicing activity (Cooper and Stevens, 1995; Shao et al., 1995), with which they liberate themselves from a precursor protein, leading to the generation of two functional and independent proteins (Gimble, 1998; Perler, 1998). The heterogeneous family of homing endonucleases can be divided into four sub-families which are characterized by one of the following sequence motifs: LAGLIDADG, GIY-YIG, H-N-H or the His–Cys box (Belfort and Perlman, 1995; Belfort and Roberts, 1997). The LAGLIDADG sequence is the most common motif and is not only found in homing endonucleases but also in other proteins interacting with nucleic acids (Dalgaard et al., 1997), e.g. HO-endonuclease (Russell et al., 1986). The intein-encoded PI-SceI from Saccharomyces cerevisiae (Gimble and Thorner, 1992, 1993) belongs to this sub-family of homing endonucleases and contains two copies of the LAGLIDADG motif. PI-SceI interacts with DNA as a monomer and recognizes an extraordinarily long DNA sequence with no apparent symmetry. Depending on the substrate, the minimal sequence that is recognized exceeds 35 bp. Whereas binding to the recognition site, which is accompanied by a distortion of the DNA, occurs also in the absence of the cofactor Mg2+, specific cleavage requires the presence of this bivalent metal ion. PI-SceI cleaves the two DNA strands in a highly concerted reaction, with no detectable accumulation of a nicked intermediate, to produce specific DNA fragments with 3′-OH and 5′-phosphate ends. Replacement of Mg2+ by Mn2+ leads to a relaxed specificity and increased rate of reaction, which is nevertheless relatively slow (Gimble and Wang, 1996; Wende et al., 1996; Grindl et al., 1998). In contrast to PI-SceI (Duan et al., 1997) and I-DmoI (Silva et al., 1999), some other members of the LAGLIDADG family are homodimers, e.g. I-CreI (Heath et al., 1997), and recognize pseudo-palindromic sequences. These homodimeric homing endonucleases contain one LAGLIDADG motif per subunit, each of which is involved in phosphodiester bond cleavage in one DNA strand. For the monomeric homing endonuclease PI-SceI, it is still a controversial issue as to whether it has one (Gimble and Stephens, 1995; Duan et al., 1997) or two active sites (Wende et al., 1996; Pingoud et al., 1998). Substitution of Asp218 or Asp326 in PI-SceI leads to complete inactivation of the nucleolytic function, a result which has been interpreted to mean that these residues together make up one single catalytic centre responsible for cleaving both strands of the DNA substrate (Gimble and Stephens, 1995), a feature without precedence among site-specific nucleases. On the other hand, the fact that the two strands are cleaved in a concerted manner argues for the existence of two catalytic centres in the monomeric enzyme (Wende et al., 1996). Support for this conjecture comes from structural comparisons. There is a significant structural similarity between I-CreI, which is a homodimer, and PI-SceI as well as I-DmoI, both of which are monomeric enzymes and have a catalytic domain with a pseudo 2-fold symmetry axis (Silva et al., 1999). Here we present evidence that PI-SceI has two catalytic centres which are responsible for the cleavage of the top and the bottom strand, respectively, of the double-stranded DNA substrate. This evidence rests on three observations, namely: (i) that PI-SceI tethered to one strand of the DNA is able to cleave both strands; (ii) that active site variants of PI-SceI exist that cleave one strand of the DNA substrate, but not, or hardly at all, the other one; and (iii) that the transition states for cleavage of the two strands are very different. Results In the following section, we present the results of different types of experiments designed to find out whether PI-SceI has one or two active sites to cleave the two strands of its DNA substrate, and, if two sites are present, to characterize these sites and to assign the cleavage events at the top and bottom strands to one or the other active site. Cleavage experiments with PI-SceI tethered to one strand of its substrate by a photocross-link If PI-SceI has only one catalytic centre to cleave the two strands of its double-stranded DNA substrate, this would mean that after cleavage of one strand the catalytic centre must undergo a major conformational transition in order to be able to attack the other strand. For an in-line attack on the phosphodiester bond, as observed for EcoRI, EcoRV, HpaII and SfiI (Connolly et al., 1984; Grasby and Connolly, 1992; Mizuuchi et al., 1999), this would literally mean that the enzyme has to ‘turn around’ its active site between the two cleavage events. To find out whether such major movements are part of the mechanism of double strand cleavage, we have tethered PI-SceI via His333 to thymine +9 in the bottom strand by photocross-linking (Pingoud et al., 1999) and analysed whether such a tethered complex is enzymatically active. As shown in Figure 1, the cross-linked PI-SceI complex is able to cleave its DNA substrate. That cleavage occurs in both strands was established by an independent experiment, in which PI-SceI was first incubated with the DNA in the presence of Mn2+ overnight in order to obtain cleavage, and then irradiated to cross-link the product to the enzyme. Cleavage followed by irradiation (which cross-links the downstream product to the protein) resulted in the same cross-linked product as irradiation (which cross-links the substrate to the protein) followed by cleavage (which releases the upstream product; data not shown). Although the kinetics of cleavage has not been analysed in detail for the tethered complex, the rate of cleavage must be similar for the free and the tethered enzyme as complete cleavage is observed by incubation overnight at room temperature in the presence of Mn2+. As His333 is located in the catalytic domain of PI-SceI (Duan, et al., 1997), this result demonstrates that the relative positions of the catalytic domain and the substrate remain largely unaltered between the two cleavage events. Figure 1.Cleavage of a radioactively labelled oligodeoxynucleotide substrate by PI-SceI tethered to its substrate as indicated at the bottom of the figure. PI-SceI was covalently linked via His333 to thymine +9 in the bottom strand by photocross-linking. The DNA in the PI-SceI–DNA complex was cleaved by incubation in the presence of Mn2+. The products of irradiation and cleavage were analysed by SDS–PAGE. (A) Silver-stained gel: lane S, molecular weight standard; lanes 1 and 2, PI-SceI–DNA complex before and after irradiation; lane 3, cross-linked PI-SceI–DNA complex after incubation with Mn2+. (B) Autoradiogram of lanes 1, 2 and 3. Download figure Download PowerPoint Cleavage experiments with PI-SceI and substrates carrying a single strand break in the scissile phosphodiester bond in the top or bottom strand If PI-SceI has two active sites for cleavage of the two strands of its double-stranded DNA substrate, it should, in principle, be possible to inactivate one catalytic centre and preserve the activity of the other. Such a PI-SceI variant, produced by site-directed mutagenesis of a presumptive catalytic residue, should be able to introduce a specific nick into one strand of a double-stranded DNA substrate and/or to cleave a DNA substrate with a specific nick in the other strand. Ideally, two sets of variants should be produced, with substitutions in one or the other catalytic centre. Obvious candidates for such substitutions are the Asp residues of the two LAGLIDADG motifs, which have been shown to be essential for catalysis in PI-SceI and related enzymes (Gimble and Stephens, 1995; Lykke-Andersen et al., 1996; Seligman et al., 1997). However, it had been shown previously that the replacement of Asp218 and Asp326 by Ala and Asn led to completely inactive enzyme variants concerning double-stranded DNA substrates (Gimble and Stephens, 1995; S.Schoettler, unpublished). In order to analyse the influence of single strand breaks in the PI-SceI cleavage positions of the top and bottom strand of the DNA substrate, cleavage experiments with fully double-stranded and nicked substrates were carried out. These experiments were performed with PI-SceI and variants carrying amino acid substitutions at catalytically important positions, other than Asp218 and Asp326. We selected Asp229, Thr341 and Lys403 because they are located close to Asp218 and Asp326 but, in contrast to these residues, do not lead to completely inactive PI-SceI variants when substituted by Asn (S.Schoettler, unpublished) or Ala (Gimble et al., 1998). As shown in Table I, a substrate with a nick at the cleavage position in the top strand is cleaved faster by wild-type PI-SceI than the intact double-stranded oligodeoxynucleotide or an oligodeoxynucleotide with a nick in the bottom strand. These data may indicate that the rate-limiting step for double strand cleavage by wild-type PI-SceI is the cleavage of the top strand, which means that the bottom strand can be cleaved more readily when the top strand is already nicked. A different result is obtained with PI-SceI variants with single amino acid substitutions in the presumptive catalytic centres (Table I). While completely inactive variants such as D218A, K301A and D326A (Gimble and Stephens, 1995; He et al., 1998) do not exhibit any cleavage activity with the nicked substrates, D229N, T341N and K403A exhibit residual activity on fully double-stranded substrates and, intriguingly, show a pronounced preference for a substrate with a nick in one strand: D229N and K403A in the top strand, T341N in the bottom strand. This suggests that Asp229 and Lys403 are involved mainly in the cleavage of the scissile phosphodiester bond in the top strand. When this bond is already cleaved, the functions of Asp229 (absent in D229N) and, to a lesser extent, of Lys403 (absent in K403A) are dispensable for the cleavage of the bottom strand. In contrast, Thr341 seems to be responsible mainly for cleavage of the bottom strand, as the T341N variant (which lacks the Thr functionality) prefers to cleave a substrate already nicked in the bottom strand. Our data suggest that the top strand is attacked first and that this is the rate-limiting step for double strand cleavage. If an oligodeoxynucleotide with a nick in the top strand is offered as a substrate, cleavage of the bottom strand is fast, presumably because of relief of conformational stress. Table 1. Cleavage of nicked substrates by PI-SceI and PI-SceI variants kapp (per h)a Double- strandedb Nicked in bottom strandb Double- strandedc Nicked in top strandc Wild-type 2.6 ± 0.03 2.7 ± 0.45 3.0 ± 0.12 9.9 ± 0.3 D326A − − − − K301A − − − − T341N + ++ + (+) D218A − − − − D229N + (+) + ++ K403A + − + + Cleavage experiments with PI-SceI variants were only evaluated in a semi-quantitative manner by measuring product concentration at single time points, taken after 30 min or 2 h, respectively: ++, wild-type activity; +, reduced activity; (+), minimal activity; −, no activity. a Apparent first order rate constant for cleavage of double-stranded and nicked substrates, obtained by assuming that a 1:1 enzyme–substrate complex is the relevant catalytic species in the single turnover experiments. b The radioactive phosphate label is at the 5′ end of the top strand. c The radioactive ddAMP label is at the 3′ end of the bottom strand. Identification of the two catalytic centres of PI-SceI by homology considerations PI-SceI has considerable structural homology with two other members of the LAGLIDADG family of homing endonucleases, namely I-CreI (Heath et al., 1997) and I-DmoI (Silva et al., 1999). The catalytic centres of these enzymes can be superimposed and corresponding residues identified in the homodimeric enzyme I-CreI and the two symmetry-related subdomains of the catalytic domain of the monomeric enzymes PI-SceI and I-DmoI (cf. Silva et al., 1999). The very good fit of these superpositions is a strong argument that these enzymes have a similar mechanism of action, which implies that the monomeric homing endonucleases PI-SceI and I-DmoI have two catalytic centres like their homodimeric relative I-CreI. The superposition of the catalytic domain of PI-SceI on the I-CreI–DNA complex allows a suggestion to be made about which presumptive catalytic amino acid residues of PI-SceI belong to which catalytic centre and are responsible for cleavage of the top and bottom strand. Figure 2 shows the superposition of the catalytic centres of I-CreI and PI-SceI together with the DNA substrate of I-CreI. In Figure 2 are also shown two Ca2+ ions from the I-CreI–DNA structure. In the hydration sphere of one Ca2+ ion, there is a water molecule that is in an ideal position for an in-line attack on the scissile phosphodiester bond of the top strand. The superposition suggests that in PI-SceI this Ca2+ ion would be bound by Asp218 and would be involved in cleavage of the top strand. Figure 2.Stereo view of the presumptive catalytic residues of PI-SceI (Brookhaven Protein Data Bank entry: 1VDE) superimposed on parts of the I-CreI–DNA co-crystal structure (Brookhaven Protein Data Bank entry: 1BP7) including the top and bottom strands of the recognition sequence (in dark and light blue, respectively), with the scissile phosphates (in yellow), the catalytic residues of I-CreI (in green) homologous to the corresponding residues in PI-SceI, two Ca2+ ions and one H2O molecule (blue cross) which is in a position to attack in-line the phosphodiester bond of the top strand. Indicated in red are Asp218, Asp229 and Lys403, and Asp326, Lys301 and Thr341 of PI-SceI, which define the two catalytic centres. Download figure Download PowerPoint Cleavage experiments with PI-SceI and phosphorothioate-substituted oligodeoxynucleotides PI-SceI recognizes an asymmetric sequence of >35 bp, with only little symmetry at the site of cleavage, i.e. ...G G T G C/G... ...C/C A C G C... Thus, one would not expect that an enzyme with only one catalytic centre makes similar base contacts with both strands at the sites of cleavage. However, for the phosphate contacts at the site of cleavage, it can be expected that the structural requirements must be similar for the two cleavage events, as the transition state involves not only the functional groups of the enzyme but also the phosphates being attacked. It is very likely, therefore, that phosphorothioate for phosphate substitutions at the site of cleavage should have a similar effect in both strands when one catalytic centre is responsible for cleavage of both strands; in contrast, when two catalytic centres are involved, one would expect differences. In order to analyse the substrate requirements of the homing endonuclease PI-SceI at and next to the site of cleavage, we have produced 12 different 47 bp oligodeoxynucleotides comprising the PI-SceI recognition sequence and containing single stereochemically pure phosphorothioate substitutions at positions located either at the cleavage position or 5′ and 3′ to this position in the top and the bottom strand, respectively. These oligodeoxynucleotides are bound as well as unmodified oligodeoxynucleotide substrates by PI-SceI (data not shown). Figure 3 shows the kinetics of cleavage of oligonucleotides modified in the top (oligodeoxynucleotides UcpR and UcpS) and the bottom strand (LcpR and LcpS), in comparison with non-modified substrates. Substitution of a non-bridging oxygen by sulfur at the site of cleavage in the top strand decreases the rate of cleavage by more than a factor of 25 in comparison with the non-modified substrate and with no stereoselectivity for the RP and SP diastereomer. In contrast, the oligodeoxynucleotide carrying the SP phosphorothioate substitution in the cleavage position in the bottom strand (LcpS) is cleaved much better than that carrying the corresponding substitution in the top strand (UcpS). The RP diastereomer (LcpR) is cleaved less efficiently (by a factor of 6) than the SP diastereomer (LcpS) (Figure 4). Figure 3.Kinetics of cleavage of non-modified (▴), RP-substituted (♦) and SP-substituted (▪) oligodeoxynucleotides, with the phosphorothioate at the cleavage position, by PI-SceI. The top strand is radioactively labelled and substituted in the panel on the left, while in the panel on the right it is the bottom strand which is radioactively labelled and substituted. Download figure Download PowerPoint Figure 4.Relative cleavage efficiencies of PI-SceI for RP and SP phosphorothioate-substituted oligodeoxynucleotide substrates. The substrates were incubated overnight with PI-SceI under standard conditions in the presence of Mn2+. U and L denote substitutions in the top and bottom strand, respectively; cp refers to the cleavage position, and 3′ and 5′ to the respective flanking positions. Download figure Download PowerPoint Sulfur substitution of a non-bridging oxygen atom, in both the RP and SP position, 3′ adjacent to the scissile phosphodiester bond in the top strand (U3′R and U3′S) does not significantly affect the extent of cleavage, demonstrating that PI-SceI does not exhibit stereoselectivity regarding the sulfur substitution at this position within the recognition sequence (Figure 4). As similar extents of cleavage were observed for the oligodeoxynucleotides U5′R and U5′S, PI-SceI also does not discriminate between the diastereomers in this position of the phosphodiester group, although the yield of product formation is 25% lower with U5′R and U5′S than with the unmodified oligodeoxynucleotide. Oligodeoxynucleotides substituted by phosphorothioate in the bottom strand at the position 5′ and 3′ to the scissile phosphodiester bond are accepted as substrates by PI-SceI. There is a 2-fold preference for L3′S over L3′R and a 3-fold preference for L5′R over L5′S (Figure 4). Thus, the most pronounced differences between the two strands regarding the effects of phosphorothioate for phosphate substitutions are observed at the position of cleavage where a precise geometry and polarity are critical for the formation of the transition state. This finding is much more compatible with a mechanism of cleavage involving two catalytic centres, which are likely to have different structural requirements regarding their substrates, than with a mechanism involving only one catalytic centre, for which one would assume that it must adopt a similar transition state for the two cleavage events. Metal ion mapping experiments with PI-SceI and an oligodeoxynucleotide substrate Divalent metal ions, such as Mg2+ or Mn2+, are obligatory cofactors for PI-SceI. They are likely to be involved directly in catalysis. In order to determine to which phosphate residues the essential divalent metal ions are bound in the PI-SceI–DNA complex, metal ion mapping experiments were performed similarly to previous descriptions (Farber and Levine, 1986; Ettner et al., 1995; Zaychikov et al., 1996). As shown for the homing endonucleases I-PorI and I-DmoI (Lykke-Andersen et al., 1997), PI-SceI accepts Fe2+ as a cofactor and displays with Fe2+ a similar activity to that seen with Mg2+ (data not shown). For the metal ion mapping experiments, the 47mer oligodeoxynucleotide was incubated in the presence or absence of PI-SceI and Fe2+/H2O2 as well as in the presence of PI-SceI and Fe2+, under conditions (low temperature, short incubation time) where hardly any cleavage occurs due to the endonuclease activity of PI-SceI. In order to determine the size of the fragments generated by hydroxyl radical attack in the metal ion mapping experiments, the 47mer was also cleaved with PI-SceI and with the restriction endonuclease AciI. In the presence of Fe2+ and H2O2, specific hydroxyl radical-induced cleavages could be detected in the PI-SceI–DNA complex but not in the free DNA (which is degraded non-specifically) (Figure 5). They occur at two major sites in the bottom strand and at three major sites in the top strand. Taking into account that hydroxyl radical-induced cleavage generates phosphate ends differing from the 5′-phosphate ends generated by PI-SceI and AciI cleavage (Hertzberg and Dervan, 1984; Stubbe and Kozarich, 1987), the sites of hydroxyl radical attack can be identified. Both strands were cleaved by hydroxyl radicals at the scissile phosphates. In the bottom strand, an additional cleavage is observed 3′ to the cleavage position, whereas for the top strand one additional cleavage occurs 5′ and one 3′ to the scissile phosphodiester bond (Figure 5C). These hydroxyl radical-induced cleavages can also be observed, albeit only as faint bands, in the absence of H2O2, as some Fe2+-induced hydroxyl radical formation takes place in H2O. The observed hydroxyl radical-induced cleavages are due to catalytically relevant metal ions as they can be suppressed by the addition of Ca2+, a specific inhibitor of the enzymatic cleavage reaction (Figure 5B). In hydroxyl radical footprinting experiments, Gimble and Stephens (1995) had observed a hypersensitive site in the top strand (presumably corresponding to site +3 in our Figure 5) and two hypersensitive sites in the bottom strand (presumably corresponding to sites −2 and −3 in our Figure 5), but did not demonstrate that these hypersensitive sites are due to Fe2+ ions specifically bound to PI-SceI. Figure 5.Metal ion mapping at the PI-SceI cleavage site. (A) Fe2+-induced hydroxyl radical cleavage of the DNA in the wt PI-SceI–oligodeoxynucleotide complex analysed by denaturating PAGE. Lanes a and b show a standard ranging from 8 to 32 nucleotides, radioactively labelled with either [γ-32P]ATP (a) or [α-32P]ddATP (b). In lanes 1-6, the results of cleavage experiments with a PI-SceI substrate labelled with [γ-32P]ATP in the top strand and in lanes 7–12 labelled with [α-32P]ddATP in the bottom strand are shown. Lanes 1 and 7, the oligodeoxynucleotide without further incubation; lanes 2 and 8, cleavage by AciI; lanes 3 and 9, cleavage by PI-SceI; lanes 4 and 10, metal ion mapping with PI-SceI; lanes 5 and 11, metal ion mapping without addition of PI-SceI; lanes 6 and 12, metal ion mapping with PI-SceI in the absence of H2O2. (B) Metal ion mapping with Ca2+ competing for Fe2+. Lanes 10 and 12 correspond to lanes 10 and 12 in (A): lane 10, metal ion mapping with PI-SceI and Mg2+; lane 12, metal ion mapping with PI-SceI in the absence of H2O2; lane 13, metal ion mapping with PI-SceI and an equimolar mixture of Ca2+ and Fe2+. (C) Sequence of the oligodeoxynucleotide used in this study. The PI-SceI cleavage positions are indicated with solid lines, the AciI cleavage positions with dotted lines. The metal ion-induced cleavages are indicated by arrows. (D) Comparison of the results of the metal ion footprinting for the wt-PI-SceI, the D326A and the D218A variant. + denotes strong hydroxyl radical cleavage and − no cleavage. Download figure Download PowerPoint In order to identify amino acid residues responsible for metal ion co-ordination, PI-SceI variants with single amino acid substitutions were used for the metal ion mapping (Figure 5D). An Asp to Ala substitution at position 326 leads to the disappearance of the hydroxyl radical-induced cleavage at position +1 in the top strand and −3 in the bottom strand, whereas the cleavage at position −2 in the bottom strand is reduced. In contrast, with the D218A variant, the hydroxyl radical cleavages at position +3 of the top strand and −2 of the bottom strand are not observed. These data indicate that hydroxyl radical formation is caused by at least two metal ions, one bound to Asp326 leading to cleavage at positions +1 in the top strand and −2 as well as −3 of the bottom strand, and the other bound to Asp218 leading to cleavage at position +3 of the top strand and −2 of the bottom strand. Gimble and Stephens (1995) had also observed that the hydroxyl radical cleavage pattern shown for the PI-SceI–DNA complex changed when substituting Asp218 and Asp326 by Ala. The hypersensitivity in the top strand of one site (presumably our site +3) disappeared in the presence of the D218A variant, and that of one site in the bottom strand (presumably our site −3) disappeared in the presence of the D326A variant. Qualitatively, their observations agree with ours, in particular that the D218A and D326A variants protect different sites in the top and bottom strand, respectively. The results can be explained by binding of two Fe2+ ions, one to Asp218 and the other to Asp326, with slight differences regarding the orientation of the two metal ions towards the two strands of the DNA substrate. Thi