DNA translocation blockage, a general mechanism of cleavage site selection by type I restriction enzymes
1999; Springer Nature; Volume: 18; Issue: 9 Linguagem: Inglês
10.1093/emboj/18.9.2638
ISSN1460-2075
AutoresPavel Janščák, Maria P. MacWilliams, Ursula Sandmeier, Valakunja Nagaraja, Thomas A. Bickle,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle4 May 1999free access DNA translocation blockage, a general mechanism of cleavage site selection by type I restriction enzymes Pavel Janscak Pavel Janscak Department of Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Switzerland Search for more papers by this author Maria P. MacWilliams Maria P. MacWilliams Microbiology and Cell Biology Department, Indian Institute of Science, Bangalore, 560 012 India Search for more papers by this author Ursula Sandmeier Ursula Sandmeier Department of Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Switzerland Search for more papers by this author Valakunja Nagaraja Valakunja Nagaraja Search for more papers by this author Thomas A. Bickle Corresponding Author Thomas A. Bickle Department of Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Switzerland Search for more papers by this author Pavel Janscak Pavel Janscak Department of Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Switzerland Search for more papers by this author Maria P. MacWilliams Maria P. MacWilliams Microbiology and Cell Biology Department, Indian Institute of Science, Bangalore, 560 012 India Search for more papers by this author Ursula Sandmeier Ursula Sandmeier Department of Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Switzerland Search for more papers by this author Valakunja Nagaraja Valakunja Nagaraja Search for more papers by this author Thomas A. Bickle Corresponding Author Thomas A. Bickle Department of Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Switzerland Search for more papers by this author Author Information Pavel Janscak1, Maria P. MacWilliams2, Ursula Sandmeier1, Valakunja Nagaraja3 and Thomas A. Bickle 1 1Department of Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Switzerland 2Microbiology and Cell Biology Department, Indian Institute of Science, Bangalore, 560 012 India 3Department of Biology, Seton Hall University, 400 South Orange Avenue, South Orange, NJ, 07079 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:2638-2647https://doi.org/10.1093/emboj/18.9.2638 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Type I restriction enzymes bind to a specific DNA sequence and subsequently translocate DNA past the complex to reach a non-specific cleavage site. We have examined several potential blocks to DNA translocation, such as positive supercoiling or a Holliday junction, for their ability to trigger DNA cleavage by type I restriction enzymes. Introduction of positive supercoiling into plasmid DNA did not have a significant effect on the rate of DNA cleavage by EcoAI endonuclease nor on the enzyme's ability to select cleavage sites randomly throughout the DNA molecule. Thus, positive supercoiling does not prevent DNA translocation. EcoR124II endonuclease cleaved DNA at Holliday junctions present on both linear and negatively supercoiled substrates. The latter substrate was cleaved by a single enzyme molecule at two sites, one on either side of the junction, consistent with a bi-directional translocation model. Linear DNA molecules with two recognition sites for endonucleases from different type I families were cut between the sites when both enzymes were added simultaneously but not when a single enzyme was added. We propose that type I restriction enzymes can track along a DNA substrate irrespective of its topology and cleave DNA at any barrier that is able to halt the translocation process. Introduction DNA translocation by proteins is a means to move along DNA and it is involved in a variety of processes such as replication (Yang et al., 1989; Kong et al., 1992), transcription (Liu and Wang, 1987), homologous recombination (Tsaneva et al., 1992), DNA repair (Koo et al., 1991; Allen et al., 1997) and DNA restriction (Yuan et al., 1980; Meisel et al., 1995). Protein tracking along DNA can occur by various mechanisms. Some proteins such as the β subunit of DNA polymerase form clamps that slide freely along DNA (Kong et al., 1992). Other proteins such as RNA polymerases or DNA helicases track along the right-handed DNA double helix and can induce changes in the secondary and tertiary DNA structure (Dröge, 1994). For the type I restriction endonucleases, DNA translocation mediates communication between the recognition and cleavage sites. These enzymes recognize specific non-palindromic DNA sequences (e.g. GAGNNNNNNNGTCA where N is any nucleotide) but subsequently make a second contact with non-specific sequences near the recognition site and pull DNA through the complex in a reaction dependent on ATP hydrolysis. DNA cleavage occurs at undefined loci that may be several thousand base pairs away from the recognition site (Rosamond et al., 1979; Yuan et al., 1980; Endlich and Linn, 1985; Szczelkun et al., 1996). Although type I restriction enzymes do not turn over in the cleavage reaction (Eskin and Linn, 1972a), the hydrolysis of ATP continues long after DNA degradation has ceased (Eskin and Linn, 1972b; Yuan et al., 1972). In addition to restriction activity, the type I restriction enzymes exhibit an N6-adenine DNA methyltransferase activity at the recognition sequence, using S-adenosyl methionine (AdoMet) as the methyl donor (Burckhardt et al., 1981). The multifunctional properties of the type I restriction enzymes are reflected in their quaternary structure. All type I restriction enzymes are composed of three different subunits: HsdS, HsdM and HsdR (Meselson and Yuan, 1968; Eskin and Linn 1972a; Suri et al., 1984; Price et al., 1987). The subunit stoichiometry of the functional endonuclease is R2M2S1 (Dryden et al., 1997; Janscak et al., 1998). The M2S1 component of this complex mediates its binding to the recognition sequence and can also function independently as a DNA methyltransferase (Taylor et al., 1992; Dryden et al., 1993; Janscak and Bickle, 1998). The HsdR subunit is essential for restriction. It contains a set of seven conserved amino acid sequence motifs typical of the helicase superfamily II that may be relevant to the ATP-dependent DNA translocation (Gorbalenya and Koonin, 1991; Murray et al., 1993). Conservative changes in any of these motifs impair both restriction and ATPase activities (Webb et al., 1996; Davies et al., 1998). The HsdM subunit contains the catalytic site for DNA methylation as well as the binding site for the methyl donor and restriction cofactor AdoMet (Willcock et al., 1994). The HsdS subunit determines DNA specificity. It contains two separate DNA-binding domains each recognizing one specific half of the recognition sequence (Fuller-Pace et al., 1984; Gubler et al., 1992). Most type I restriction–modification systems characterized so far are from enterobacteria. Based on subunit complementation, DNA hybridization and antibody cross-reactivity experiments, these systems are grouped into four families (Murray et al., 1982; Price et al., 1987; Titheradge et al., 1996). Members of the same family can interchange individual subunits, but members from different families cannot. Although the genetic complementation is seen solely within a family, it is probable that all families share common reaction mechanisms for both DNA methylation and DNA restriction: (i) the predicted amino acid sequences of the products of all hsdR genes so far sequenced contain the helicase-like domain and a number of other short conserved regions that may be implicated in DNA restriction (Titheradge et al., 1996); (ii) amino acid sequence comparison and tertiary structure modelling suggest a common structure for all type I DNA methyltransferases (Dryden et al., 1995); and (iii) the biochemical properties of the enzymes studied are quite similar. The mechanism by which type I restriction enzymes select cleavage sites is not clearly understood. The enzyme activity depends on the nature of the DNA substrate. Linear DNA molecules with a single recognition site are either refractory to cleavage (Rosamond et al., 1979; Dreier et al., 1996) or undergo limited cleavage at a very high excess of enzyme over DNA (Murray et al., 1973; Szczelkun et al., 1996). However, linear DNA molecules containing two or more sites are good substrates for cleavage. The cleavage of these molecules essentially occurs in the region between the recognition sites, and the enzyme shows a preference for cleavage roughly half way between the sites (Studier and Bandyopadhyay, 1988; Dreier et al., 1996; Szczelkun et al., 1997). Additional preferred cleavage sites located in the vicinity of the recognition sites are also observed with some enzymes (Szczelkun et al., 1997). Varying the relative orientation of two asymmetric recognition sequences does not affect the cleavage (Dreier et al., 1996; Szczelkun et al., 1997). On the basis of these findings, a cooperative model has been proposed according to which a type I restriction enzyme bound to its recognition site translocates DNA towards itself simultaneously from both directions and DNA cleavage occurs at the site where two convergently translocating enzyme molecules meet (Studier and Bandyopadhyay, 1988; Dreier et al., 1996). The cleavage of one-site substrates has been suggested to be a result of collision between an enzyme tracking from the recognition site and a second enzyme bound to a non-specific site (Studier and Bandyopadhyay, 1988). In contrast to linear substrates, circular DNA molecules containing one recognition site are cleaved efficiently (Rosamond et al., 1979; Dreier et al., 1996; Janscak et al., 1996). In one model, it is proposed that this susceptibility is a consequence of changes in DNA topology induced by enzyme tracking along the major or the minor groove of the DNA helix (Szczelkun et al., 1996). It is hypothesized that the translocation of circular DNA past the enzyme molecule anchored to the recognition site leads to generation of overwound DNA in the contracting domain and underwound DNA in the expanding domain. The accumulation of torsionally stressed DNA ultimately would stall enzyme translocation and result in DNA cleavage (Szczelkun et al., 1996). To investigate further the link between DNA translocation and the cleavage event, we examined the action of type I restriction enzymes on DNA molecules that contained potential blocks to DNA translocation such as positive supercoils or Holliday junctions. We also investigated interactions between two type I restriction enzymes from different families. The results from these experiments suggest that type I restriction enzymes can translocate DNA irrespective of its topological status and have the potential to cleave DNA at barriers that are able to halt the DNA translocation process. Results Cleavage of positively supercoiled DNA by EcoAI A build up of positive supercoils in the contracting DNA loop has been proposed to be the trigger for DNA cleavage by type I restriction enzymes on circular substrates, by causing either a halt or a pause in the helix-tracking process, during which the enzyme cuts both DNA strands (Szczelkun et al., 1996). To address this model, we have investigated cleavage of positively supercoiled plasmid DNA by EcoAI (IB family). If positive supercoiling presented a barrier for tracking, a positively supercoiled DNA substrate would be expected to be cleaved faster than a relaxed substrate, and cleavage sites would be located near the enzyme recognition site. Positive supercoils were introduced into pJP25 DNA, a 2.87 kb plasmid containing a single EcoAI recognition site, as described in Materials and methods. We found that this DNA preparation was cleaved readily by EcoAI to linear DNA with an initial rate slightly lower than that observed with relaxed molecules, but slightly higher than the rate of cleavage of negatively supercoiled molecules (Figure 1). To determine the position of double-strand breaks introduced by EcoAI into the positively supercoiled, relaxed and negatively supercoiled substrates, the corresponding EcoAI linear products were digested with XmnI, which has a unique site in pJP25 located 970 bp away from the EcoAI site. All three subsequent XmnI digestions appeared as uniform smears of DNA on an agarose gel (Figure 2). This shows that EcoAI cleaved all three substrates at random positions throughout the DNA circle. Thus, it appears that neither positive nor negative supercoiling presents a block to DNA translocation by type I restriction enzymes. The observed differences in cleavage rates (Figure 1) may reflect different numbers of DNA supercoils in individual substrates since better resolution of DNA topoisomers on a long agarose gel (not shown) revealed that the positively supercoiled pJP25 molecules had a lower degree of supercoiling than the negatively supercoiled pJP25 molecules from HB101. These results agree with previously published data demonstrating that EcoR124I endonuclease activity increases with a decreased number of negative supercoils in a DNA substrate (Janscak et al., 1996). Thus, it seems that both negative and positive supercoils reduce the rate of DNA cleavage by type I restriction enzymes relative to relaxed substrates. Figure 1.Kinetics of DNA cleavage by EcoAI on positively supercoiled, relaxed and negatively supercoiled plasmid substrates. (A) EcoAI restriction assay. The negatively supercoiled (−SC) pJP25 DNA (single EcoAI site), isolated from E.coli HB101, was converted to the relaxed and positively supercoiled (+SC) forms, respectively, as described in Materials and methods. Cleavage reactions were carried out in buffer C at 37°C and contained 15 nM DNA and 15 nM EcoAI endonuclease. Aliquots were removed at the indicated times and analysed on a 0.9% agarose gel run in 0.5× TBE buffer at 1.5 V/cm for 12 h. DNA was visualized by ethidium bromide staining. The positions of supercoiled (SC), linear (L), relaxed (R) and nicked (NC) forms of plasmid DNA are marked on the left of the gel. The additional bands in the relaxed DNA lanes correspond to topoisomers with a low degree of negative supercoiling resulting from incomplete relaxation of pJP25. (B) Plot of the relative intensity of the linear DNA bands on the agarose gel shown in (B) against time for cleavage of positively supercoiled (○), relaxed (▵) and negatively supercoiled (□) substrates by EcoAI. The gel was quantified by densitometric scanning. The relative intensity of linear DNA bands is expressed as a percentage of total DNA per lane. Download figure Download PowerPoint Figure 2.Location of EcoAI cleavage sites on positively supercoiled, relaxed and negatively supercoiled DNA substrates. Negatively supercoiled (−SC) pJP25 DNA (a single EcoAI site), isolated from E.coli HB101, was converted to the relaxed and positively supercoiled (+SC) forms, as described in Materials and methods. The 20 μl cleavage reactions were carried out in buffer C at 37°C and contained 15 nM DNA and 80 nM EcoAI. Following a 10 min incubation, the reactions were stopped by heating at 70°C for 15 min and divided into two aliquots. One aliquot was treated further with XmnI (unique site in pJP25) for 30 min. Samples were analysed by agarose gel electrophoresis as described in Figure 1. The presence of restriction enzymes in individual reactions is indicated by (+) above each lane. The positions of supercoiled (SC), linear (L), relaxed (R) and nicked (NC) forms of plasmid DNA are indicated on the right of the gel. The appearance of a uniform smear of DNA in the XmnI lanes indicates that EcoAI cleavage occurred at random positions throughout the DNA circle. Since EcoAI did not cleave either substrate completely, the subsequent XmnI digestions also contain the full-size linear form of pJP25. Download figure Download PowerPoint Cleavage of DNA substrates containing a Holliday junction by EcoR124II To examine the effect of physical blocks to DNA translocation on DNA cleavage by type I restriction enzymes, we used DNA molecules containing a Holliday junction generated in vivo by Xer site-specific recombination of pSD115, a 4.95 kb plasmid which carries two directly repeated cer recombination sites and a single site for the type IC restriction endonuclease EcoR124II. A stable intermediate of this reaction (figure-of-eight structure DNA) is composed of 2.6 and 2.35 kb circular duplexes joined by a Holliday junction that results from incomplete strand exchange at the cer sites (McCulloch et al., 1994). The Holliday junction is confined to a region of ∼290 bp of homology (cer sites) through which the junction can migrate spontaneously (McCulloch et al., 1994). The pSD115 plasmid preparation from induced cells of Escherichia coli RM40 contains a mixture of several species: 4.95 kb (SC) substrate, non-catenated and catenated figure-of-eight intermediates, and the two final circular recombination products with sizes of 2.6 and 2.35 kb. On an agarose gel, the catenated figure-of-eight molecules co-migrate with 4.95 kb (SC) pSD115 DNA, while non-catenated molecules migrate slightly faster (Zerbib et al., 1997). Cleavage of the figure-of-eight DNA molecules in one or both supercoiled domains with type II restriction enzyme(s) results in DNA structures which were easily separated from other pSD115 digestion products on an agarose gel (not shown) and could be purified and used as substrates for EcoR124II. EcoRI cleavage of the figure-of-eight molecules (unique site in pSD115) resulted in an α-structure with the EcoR124II recognition site situated in the 2.35 kb supercoiled domain (Figure 3A). In contrast to the random location of cleavage sites on circular substrates, the α-structure was found to be cleaved by EcoR124II into two discrete DNA fragments with sizes similar to the sizes of the linearized forms of the final Xer recombination products which are 2.35 and 2.6 kb (Figure 3B, bands P1 and P2). This indicated that EcoR124II cleaved the 2.35 kb domain at two sites, one on either side of the Holliday junction, consistent with the bi-directional translocation model (Studier and Bandyopadhyay, 1988). The α-structure was refractory to cleavage by the EcoR124I endonuclease which has no recognition site in pSD115 (not shown). It is worth noting that the α-structure substrate gave two closely migrating bands on agarose gels run in the presence of ethidium bromide (Figure 3B). It was shown previously that superhelical torque induced by ethidium bromide binding to a circular DNA can force a Holliday junction to locate at either of two possible extreme positions within a region of homology (Yang et al., 1998). For the α-structure, this would result in two species with different arm length ratios and hence slightly different mobilities. A time course of α-structure digestion by EcoR124II revealed a reaction with two intermediates being formed (Figure 3B). The first intermediate to appear was nicked α-structure (band I1), which agrees with observations on circular DNA substrates (Janscak et al., 1996). In the second reaction step, a presumably branched DNA molecule was produced by a single double-strand break on one side of the junction (band I2). Since the first double-strand break can occur on either side of the Holliday junction (bi-directional translocation) and anywhere within the region of homology (branch migration), the second reaction intermediate appeared as a broad band on agarose gel corresponding to a population of branched intermediates with different lengths of arms and therefore different electrophoretic mobilities. A higher intensity at the bottom of the smeared band suggested a preferred position for cleavage. Finally, the branched structure was resolved to the two linear products by a cleavage on the other side of the junction, perhaps by introduction of another double-strand break or just by nicking the continuous strand. Figure 3.EcoR124II cleavage of α-structure DNA containing a single EcoR124II site in the supercoiled domain. (A) Diagram of the α-structure resulting from EcoRI cleavage of the figure-of-eight molecules generated by Xer recombination of pSD115. The single EcoR124II recognition site is located in the 2.35 kb supercoiled domain. The sum of lengths of the two linear arms (2.6 kb) is indicated above the diagram. The 290 bp regions of homology (cer) are shown in grey. The orientation of the cer recombination sites is indicated by black arrowheads. For clarity, the diagram is not drawn to scale. The Holliday junction is drawn in parallel configuration. (B) EcoR124II restriction time course. Reactions were carried out at 37°C in buffer C and contained 10 nM DNA and 30 nM EcoR124II. Aliquots were removed at the indicated time points and analysed by electrophoresis on a 1% agarose gel run in 0.5× TBE buffer, containing ethidium bromide (0.5 μg/ml), at 2.5 V/cm for 12 h. The positions of the α-structure DNA, EcoR124II products (P1, P2) and reaction intermediates (I1, I2) are shown to the right of the gel. As a control, total plasmid DNA isolated from induced RM40/pSD115 was cleaved with EcoRI and EcoRV (lane C). This converted the Xer recombination products to their linear forms with sizes of 2.35 and 2.6 kb indicated to the right of the gel. Lane S contains the supercoiled form of pSD115 and lane M contains DNA size markers. Download figure Download PowerPoint The figure-of-eight DNA molecules were found to be cleaved by EcoR124II at the same positions as the α-structure when assayed for cleavage in the mixture with the other DNA species produced by Xer recombination (not shown). To investigate whether EcoR124II endonuclease can cut DNA at a Holliday junction present on linear substrates, the figure-of-eight DNA molecules were treated with EcoRI and EcoRV (unique sites in pSD115) to produce a χ-structure with the EcoR124II site located in one arm (Figure 4A). In contrast to a linear DNA with a single recognition site, this DNA substrate was cleaved by EcoR124II to result in two discrete DNA fragments (Figure 4B, bands P1 and P2). The sizes of these fragments and their restriction digest profiles (not shown) suggested that the cleavage of the χ-structure occurred at the Holliday junction, thereby releasing the arm containing the EcoR124II site. Interestingly, this fragment was approximately the same size as the 2.14 kb linear fragment produced from cleavage of the χ-structure with MluI, which has a single recognition site at the distal end of the cer sites (Figure 4A). This suggests that EcoR124II promoted branch migration to the end of the region of 290 bp homology and then introduced a double-strand break at the site where the further branch migration was blocked by DNA heterology. In addition, the rate of cleavage of the χ-structure by EcoR124II was much lower than that of the α-structure with an EcoR124II site in the supercoiled domain (Figure 3B). This could mean that the Holliday junction may not completely block DNA translocation by EcoR124II and some fraction of enzyme molecules could track across the junction via the continuous strand and dissociate from non-specific DNA when they encounter the DNA end. Figure 4.Cleavage of χ-structure DNA by EcoR124II. (A) Diagram of the χ-structure resulting from EcoRI–EcoRV digestion of the figure-of-eight molecules generated by Xer recombination of pSD115. The 290 bp regions of homology (cer) are shown in grey. The orientation of the cer recombination sites is indicated by black arrowheads. The unique EcoR124II recognition site is located in the 2.14 kb arm. For clarity, the diagram is not drawn to scale. The Holliday junction is drawn in parallel configuration. (B) EcoR124II restriction time course. Reactions were carried out and analysed as described in Figure 3. Reaction time points are indicated above each lane. The positions of the χ-structure and the two EcoR124II products (P1, P2) are indicated on the right of the gel. Two additional, faint bands appear on the gel. The faster migrating band corresponds to the EcoRI–EcoRV fragment of pSD115 that was not completely removed by gel isolation of the χ-substrate (not cleaved by EcoR124II). The slower migrating band could correspond to a variant of the χ-structure with the branch point located at a different position. As a control, the χ-structure was cleaved with MluI (lane indicated by MluI). The positions of the 2.14 and 2.33 kb linear products of the control reaction, resulting from dissociation of the originally produced χ-structure due to spontaneous branch migration, are indicated on the right of the gel. The other two fragments produced by MluI run off the gel. Lane M contains DNA size markers. Download figure Download PowerPoint Cooperation between two type I restriction enzymes from different families in cleavage of linear DNA The experiments with Holliday junction substrates demonstrated that a physical block to DNA translocation can trigger DNA cleavage by a type I restriction enzyme. This suggests that cleavage of linear DNA with two recognition sites does not occur through specific protein–protein contacts between two translocating enzyme molecules. Instead, prevention of DNA translocation by collision between the two translocating enzymes is the trigger for DNA cleavage. We tested this prediction by examining the consequence of convergent DNA translocation by two type I restriction enzymes from different families, which show little amino acid homology. Linear DNA substrates containing a single site for each enzyme were used. Such DNA substrates are refractory to cleavage if only one of the enzymes is present. It should be noted that cooperation between EcoR124II and EcoDXXI, two type IC family enzymes, in DNA cleavage was demonstrated previously (Dreier et al., 1996). However, the subunits of these enzymes show a high level of amino acid identity and are also interchangeable. We investigated combinations of EcoKI (IA family) with either EcoAI (IB family) or EcoR124I (IC family). Plasmids pJP25 (EcoKI + EcoAI) and pJP39 (EcoKI + EcoR124I) were cut by the type II restriction enzyme AlwNI to produce 2.9 kb linear substrates with type I recognition sites in tail-to-tail orientation (Figure 5A). The distance between the sites in both preparations was ∼0.9 kb. DNA substrates were treated for 10 min with a 7-fold molar excess of appropriate enzymes, and the appearance of cleavage products was monitored by agarose gel electrophoresis (Figure 5B). Under these reaction conditions, neither substrate was digested if only one of the corresponding enzymes was present. In contrast, when both enzymes were added, DNA cleavage occurred. The cleavage produced by the EcoKI–EcoAI mixture was more efficient than that produced by the EcoKI–EcoR124I mixture (Figure 5B). In both cases, agarose gels revealed not only the smear of heterologous DNA fragments from random cleavage events, but also a series of discrete bands within the DNA smear, indicating preferred cleavage sites (Figure 5B). To identify the discrete products, the gels were scanned by densitometry (Figure 5C). The sizes of individual fragments were determined from the position of the corresponding peaks on the x-axis of the densitogram relative to DNA size markers. This allowed us to determine approximate boundaries for the major region of cleavage. For the EcoKI–EcoAI mixture, the sizes of discrete fragments suggested that the majority of cleavage events occurred within the region starting half way between the sites and ending at the EcoKI site. For the EcoKI–EcoR124I mixture, the majority of cleavage events occurred in a short region near the EcoR124I site. Figure 5.Cooperation between two type I restriction enzymes from different families in cleavage of linear DNA. (A) Diagrams of DNA substrates. Plasmids pJP25 and pJP39 were cleaved with AlwNI to produce substrates lin-AK and lin-RK, respectively. DNA is represented as a thin rectangle. The positions and orientations of asymmetric EcoKI (K), EcoAI (A) and EcoR124I (R124) recognition sites are shown by filled arrowheads. The numbering of the sites refers to the position of the first base pair of the recognition sequence. (B) Restriction assay. Reactions were carried out at 37°C in buffer C. The enzymes (100 nM) were added to the appropriate DNA substrate (14 nM) individually or in combination. Following an 8 min incubation, aliquots were analysed by electrophoresis on a 1% agarose gel run in 0.5× TBE buffer, containing ethidium bromide (0.5 μg/ml), at 3 V/cm for 4 h. (C) Densitometric scans of the gels shown in (B). The sizes of fragments which correspond to major peaks of the densitometric traces are indicated. The regions encompassing the preferred cleavage sites on individual substrates are shown on the DNA representations in (A) as grey boxes. Download figure Download PowerPoint Discussion Type I restriction enzymes specifically bind to their DNA recognition sites, but cleave DNA at variable distances from their recognition sites. The enzyme molecule reaches its non-specific cleavage site by translocation of DNA via a secondary contact site while remaining fixed to the recognition site. The mechanism by which a type I restriction enzyme selects its cleavage site has not been identified unequivocally. In one model for cleavage site selection, DNA cleavage occurs at the site where two convergently translocating enzyme molecules meet (Studier and Bandyopadhyay, 1988). According to this model, cooperation between the two enzyme molecules is required for DNA cleavage. Another model invokes as the c
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