Mol Gen Genet (1998) 258: 488±493
Ó Springer-Verlag 1998
ORIGINAL PAPER
P. Polaczek á K. Kwan á J. L. Campbell
GATC motifs may alter the conformation of DNA depending on sequence context and N6-adenine methylation status: possible implications for DNA-protein recognition Received: 16 December 1997 / Accepted: 24 February 1998
Abstract As part of our analysis of the role of a uniquely clustered set of dam methylation sites (the motif GATC) within the origin of DNA replication in Escherichia coli, we have studied the eect of GATCs in various methylation states on the intrinsic curvature of DNA. We have designed a set of DNA linkers and used commercially available linkers containing GATC motifs. The linkers were ligated and the electrophoretic mobility of the resulting multimers in dierent states of methylation was tested relative to reference fragments. We report that properly phased GATCs in certain sequence environments modulate DNA curvature and that these eects may be enhanced by N6-adenine methylation of the GATCs. These structural alterations may in turn aect DNA-protein interactions, especially those involving proteins that rely on both primary sequence and structure for recognition. We present an example, where introduction of a GATC within an integration host factor (IHF) binding site, which does not alter the consensus sequence, reduces the binding anity of the protein for the modi®ed site. Key words GATC á dam methylation á DNA curvature á IHF á Escherichia coli
Introduction Methylation of adenine and cytosine residues represents the principal form of DNA modi®cation. N6-adenine methylation is limited to enteric bacteria and is implicated in important cellular processes: methyl-directed repair (Modrich 1989), replication (Crooke 1995; Slater Communicated by R. Devoret P. Polaczek á K. Kwan á J. L. Campbell (&) Braun Laboratories 147-75, California Institute of Technology, Pasadena, California 91125, USA Fax: +626-405-9452; e-mail:
[email protected]
et al. 1995) and gene expression (Roberts et al. 1985; Huisman and de Graaf 1995). Methylation of adenine residues occurs at symmetric GATC sites and is catalyzed by the dam gene product (DNA adenine methyltransferase). Many eects of dam methylation on cellular functions are well documented but the molecular basis for these eects is not well understood. Methylation has been shown to lower the thermodynamic stability of GATCs (Engel and von Hippel 1974, 1978; Fazakerley et al. 1985) and to introduce slight curvature as judged by the electrophoretic migration of BamHI (GGATCC) linker multimers (Diekmann 1987). Methylation was also shown to enhance intrinsic as well as protein-induced curvature (Kimura et al. 1989; Polaczek et al. 1997). The presence of GATCs in conserved regulatory regions of the genome is intriguing. In the Escherichia coli minimal origin of replication (oriC) 11 GATC sites are located within a stretch of 245 bp (Zyskind et al. 1983). In contrast, in most bacteriophages GATC sites are underrepresented (none are present in the 5386-bp genome of /X174; Blaisdell et al. 1996; Henault et al. 1996). During the course of a study on the interactions of IHF at E. coli oriC, we noted signi®cant alterations in the structure of the origin depending on the methylation status of the DNA template (Polaczek et al. 1997). We concluded that N6-adenine methylation contributes to the overall architecture of the origin and suggested a positive role for dam methylation in initiation of DNA replication in E. coli as an element required for proper initiation complex assembly. The IHF site at oriC (Polaczek 1990) is conserved in enteric bacteria and, unlike other known IHF sites, the oriC site contains three GATCs, each located at a position that contacts IHF. Methylation at these sites leads to a modest increase in the anity of IHF for its site, but its major eect is to enhance the IHF-induced bend at the origin (Polaczek et al. 1997). We proposed that methylation has at least a dual role as a positive regulator of DNA replication: in¯uencing the architecture of
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the IHF/oriC complex and lowering the helix stability to promote unwinding of the origin sequences. Here, as an extension of the oriC studies, we address the question of how GATC sequence motifs alter the physical properties of selected DNA fragments and the eect of adenine methylation of GATCs in dierent sequence environments. We have also tested the idea that structural alterations imposed by the presence of GATCs in certain sequence contexts may alter DNAprotein interactions.
Materials and methods Synthetic linker ligation assay The synthetic linker ligation assays were performed as described previously (Koo et al. 1996). Brie¯y, linkers were 5¢- end labeled with [c-32P]ATP and polynucleotide kinase, followed by a cold ATP chase. Two complementary oligonucleotides were mixed, heated to 55° C and cooled slowly to 0° C. T4 DNA ligase (New England Biolabs) was added to the hybridized mixture and ligation was allowed to proceed overnight. Ligations of self-complementary linkers were performed according to the protocols provided by the manufacturer (New England Biolabs). BamHI linkers (CGGGATCCCG) were used as reference standards. Some of the ligated products were methylated with Dam methylase (New England Biolabs) in vitro. The methylation status of the fragments was veri®ed (Polaczek et al. 1997) by assaying dierential susceptibility to the restriction enzymes MboI and DpnI (New England Biolabs). In oligonucleotides containing the IHF site (see Fig. 3), the methylation status of the leftmost GATC is uncertain because resistance to MboI cleavage may either be a result of methylation or of the positioning of this site close to the end of the fragment. The products were run on 8% polyacrylamide gels in 1 ´ TBE (10 ´ TBE is 1 M TRIS-HCl pH 8.0, 1 M boric acid, 0.01 M EDTA) buer at room temperature with a voltage gradient of 7.5 V/cm and the gels were autoradiographed. Gel mobility shift analysis Oligonucleotide fragments were end labeled and annealed as described above. The labeled fragments were puri®ed from 5% acrylamide gels (acrylamide:bis-accrylamide 30:1). The experimental conditions for gel shift assays were described previously (Polaczek 1990). Approximately 0.1 pmol of each of the end-labeled restriction fragments was incubated with increasing amounts of IHF protein. The reaction buer contained 50 mM TRIS-HCl pH 7.4, 70 mM KCl, 10% (v/v) glycerol, 1 mM EDTA, 1 mM b-mercaptoethanol, 7 mM MgCl2, 3 mM CaCl2, 200 mg/ml BSA. Poly(dI-dC) á poly(dI-dC) synthetic polymer (Pharmacia) was used as a nonspeci®c competitor at a ®nal concentration of 50 lg/ml. The reaction mixtures were incubated for 20 min at 25° C and resolved on 5% polyacrylamide gels (monomer:bis 30:1) by electrophoresis in 0.5 ´ TBE at 4° C. Following electrophoresis gels were autoradiographed.
Results Electrophoretic migration of GATC-containing linker multimers in dierent states of methylation Linker ligation assays oer a sensitive method for assessing structural changes in DNA imposed by the presence of speci®c sequence elements. In this report a
series of model DNA templates was constructed to document how GATC sequence motifs in various methylation states and in various sequence contexts alter the physical properties of DNA fragments. First, we compared the mobility of A-tract linker multimers (GGCCA5CT)n with A-tract multimers containing phased GATCs (GATCA5CT)n. A-tract runs are the principal sequence elements responsible for DNA curvature (Koo et al. 1986). As shown in Fig. 1 (panels 1 and 2) and quanti®ed in Fig. 2, the (GGCCA5CT)n ligamers (panel 2, lane c), like a similar sequence studied previously (GGCCA5CG)n (Koo et al. 1986; panel 1, lane a), were signi®cantly retarded as compared to the unbent reference DNA (lanes R) of the same length (the BamHI reference linkers are described in Materials and methods). Retardation of the (GGCCA5CG)n oligomer is presumably due to A-tract induced curvature (Koo et al. 1986). The electrophoretic mobility of the GATC¯anked A-tract multimers (GATCA5CT)n, was only slightly faster than the GGCC-¯anked A tract, (GGCCA5CT)n, however (Fig. 1, panel 2, lanes b and c). Since A-tract induced curvature is known to be markedly aected by neighboring sequence elements (Haran et al. 1994), the more relevant comparison is between the mobility of methylated and unmethylated GATC oligomers (Fig. 1, panel 1, lanes b and c). In vitro methylation of GATC-containing polylinkers with Dam methyltransferase signi®cantly increased the mobility of the oligomers as compared to their unmethylated counterparts. This may imply that GATC methylation-induced curvature towards the minor groove is out of phase with the minor groove of curved A tracts or, alternatively, that GATCs modulate the structure of neighboring A tracts. Homopolymeric oligo (dC á dG) tracts are expected to be conformationally rigid. This rigidity is attributed to the three hydrogen bonds in each consecutive base pair in conjunction with a sterically compact minor groove with close van der Waals contacts, a strong stacking free energy, and an altered electrostatic potential. It was reported recently, however, that C tracts (C5N6)n properly phased with the helical repeat appeared to cause slight DNA curvature (Biburger et al. 1994), in contrast to earlier studies in which no detectable curvature of phased C-tract ligamers (C5N5)n was detected (Koo et al. 1986). We noted that the C-tract ligamers used in the studies of Biburger et al. (1994) where curvature was observed contained phased GATC sites (GATCCCCCTCT)n. To address the question of whether the altered mobility was due to the presence of GATCs in the C tracts, we synthesized oligonucleotides with a single purine substitution at the second position (A to G) of the GATC sequence. As shown in Fig. 1, the electrophoretic mobility of this ligamer was normal compared to the reference DNA. Ligamers containing the C-tracts and GATC sites migrated more slowly. Methylation of the internal adenines of the GATCs further reduced mobility. Our results suggest that the curvature in C tracts observed previously (Biburger et al.
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Fig. 1 Electrophoretic mobility of GATC-containing linker multimers in dierent sequence contexts and dierent methylation states. Lane R contains BamHI linker multimers that serve as reference standards. Panel 1. Lane a, (GGCCA5CG)n; lane b, (GATCA5CT)n unmethylated; lane c, (GATCA5CT)n methylated; lane d, same as a. Panel 2. Lane a, (GGCCA5CG)n; lane b, (GATCA5CT)n unmethylated; lane c, (GGCCA5CT)n. Panel 3. Lane a, (GGTC5TCT)n; lane b, (GATC5TCT)n unmethylated; lane c, (GATC5TCT)n methylated; lane d, same as a. Panel 4. Lane a, (ATCGATCGAT)n methylated; lane b, (ATCGATCGAT)n unmethylated. Panel 5. Lane a, (TCGCGATCGCGA)n methylated; lane b, (TCGCGATCGCGA)n unmethylated. The dots indicate migration of 110-bp fragments, except in Panel 5, PvuI(NruI), where the lower dots indicate 60-bp and the upper dots 120-bp fragments. Note that PvuI (NruI) linkers in panel 5 are 12mers, other sequences tested are multimers of 11mers and the BamHI linkers are 10mers (CGGGATCCCG)
1994) is likely to be a consequence of phased GATCs in the C-rich environment, further enhanced by adenine methylation at these sites. Interestingly, the control DNA fragments used in the studies of Biburger et al. (1994) also contained phased GATCs in a dierent sequence context (GATCCTCTGAT)n, but showed apparently normal gel mobility. Multimers of the BamHI endonulease recognition sequence with the composition (CGGGATCCCG)n have the same electrophoretic mobility as DNA of random sequence and therefore are often used as reference samples in gel mobility shift assays such as those shown in Figs. 1 and 2 (Koo et al. 1986; McNamara and Harrington 1991; Biburger et al. 1994). Methylation of the internal GATC sequence was previously shown to
reduce the mobility of BamHI ligamers slightly (Diekmann 1987). We have con®rmed these results (data not shown). We note that in BamHI multimers (CGGGATCCCG)n the GC-rich environment of GATCs contains phased CCGCGG sequences, which have been shown to have an unusual structure (Malinina et al. 1994). In the PvuI (ClaI) linker multimers (ATCGATCGAT)n, both unmethylated and methylated DNA fragments show reduced gel mobility relative to the reference fragments, but methylation of the phased GATCs causes a slight increase in gel mobility relative to the unmethylated fragment (Fig. 1, panel 4; Fig. 2, bottom graph). Thus, in this particular sequence environment the eect of methylation is opposite to the one observed with C-tract or BamHI multimers. We next tested the electrophoretic mobility of a PvuI (NruI) linker multimer (TCGCGATCGCGA)n, which has two GATC sites within a single turn of the helix. Methylation of the GATC sites in this ligamer had no eect on gel mobility (Fig. 1, panel 5). Positioning of GATCs in IHF binding sites When two GATCs are positioned within one turn of the helix, as in the case of PvuI (NruI) linkers (GATCGCGATC)n, the electrophoretic mobility of such multimers is normal relative to standard linker multimers, and is independent of the methylation state (see the previous section of Results). This may suggest that
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perfect match to the oriC site with respect to the conserved arms of the core consensus sequence, IHF does not bind to this site. The IHF site in the dnaA promoter region has a GATC in the nonconserved 4-bp spacer region between the two IHF ``half-sites''. This places two GATCs within one turn of the helix ± on opposite faces ± possibly creating an S-shaped molecule (GATCAAGATC) to which IHF cannot bind. It is interesting to note that there is a strong bias in the distribution of GATC motifs in the E. coli genome and pairs such as GATCNNGATC are underrepresented (Henault et al. 1996). IHF has been shown to bind preferentially to curved DNA in its non-speci®c interactions with DNA (Bonnefoy and Rouviere-Yaniv 1991), suggesting that the binding anity of the protein is dependent on DNA structure. To test whether lack of IHF binding in the dnaA promoter region is caused by the out-of-phase GATCs or is attributable to ¯anking sequences, we used two double-stranded oligomers, one matching the IHF binding site at oriC and the other carrying a GATC substitution in the spacer region. As shown in Fig. 3, IHF binding to the mutant oriC site is signi®cantly reduced. At high concentrations of IHF only a faint band representing DNA/IHF complex can be observed with unmethylated mutant fragment (Fig. 3, lane k). The lack of apparent eects of methylation on binding, such as those previously observed with oriC restriction fragments (Polaczek et al. 1997), is likely to be due to the use of short oligonucleotides and the consequent lower stability of the complexes formed (Bonnefoy and RouviereYaniv 1992). Mixing experiments using fragments of dierent lengths in the presence of speci®c competitors are required to establish conclusively the eects of methylation on IHF binding, as we have previously shown (Polaczek et al. 1997).
Discussion
Fig. 2 Graphic representation of the electrophoretic mobility data. The ratio (RL) of apparent length of each multimer to the actual length of ligated BamHI linker multimers, serving as reference size standards is plotted against the latter parameter. Non-bent DNA fragments of normal electrophoretic mobility have an RL value of 1.0. RL values above 1.0 indicate anomalous mobility due to DNA bending
the two out of phase GATCs eectively cancel each other with respect to curvature. We had previously identi®ed a putative IHF site in the dnaA promoter region (Polaczek 1990). Despite a
Our results demonstrate that GATCs may aect DNA structure in dierent ways depending on the sequence context and the state of methylation. Computational analysis suggested that several dinucleotide steps other than AA/TT (present in A tracts) may cause de¯ections in the helical axis of DNA and contribute to DNA curvature (Bolshoy et al. 1991). This type of analysis may, in general, correctly predict DNA conformation, although discrepancies due to sequence contexts do exist (Dlakic and Harrington 1996). Strong modulatory properties of a CA dinucleotide step on DNA curvature have been observed (Bolshoy et al. 1991; Lyubchenko et al. 1993; Nagaich et al. 1994). Phased CA/TG elements enhance the eciency of cyclization, but two CA/ TG elements separated by one-half helical turn compensate for each other and reduce the cyclization eciency, suggesting anisotropic ¯exibility (Lyubchenko et al. 1993). Similar compensatory mechanisms may account for the lack of eects of methylation on the
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Fig. 3 Binding of IHF to double-stranded synthetic oligonucleotides containing oriC sequences at the IHF site (shown as IHF oriC) and to a mutant IHF site containing an additional GATC site (underlined) in the nonconserved spacer region of the IHF recognition sequence (shown as IHF oriC/dnaA). The mutant IHF site is identical to the putative IHF site in the dnaA promoter region with respect to the consensus sequence but has oriC ¯anking sequences. Me+ and Me) denote the methylated or unmethylated status of the oligonucleotides, respectively. The methylation status of the leftmost GATC site (at the 5¢-end) was not veri®ed (see Materials and methods). IHF concentrations are as follows: lanes a and n contain no protein; lanes b, e, h and k contain 75 ng of IHF; lanes c, f, i and l50 ng; and lanes d, g, j and m 37.5 ng. At the bottom a comparison of IHF sites at oriC and dnaA with the IHF consensus recognition sequence derived from studies of Gardner and Nash (1986) is shown (upper case, bold); the IHF site spacer and ¯anking sequences are printed in lower case
mobility of PvuI (NruI) linker multimers, as shown here, and may suggest that the two out-of-phase GATCs effectively cancel each other with respect to their eects on curvature. The IHF site in the dnaA promoter region, which is identical to a site in oriC with respect to conserved bases, is nonfunctional (Polaczek 1990). Based on this observation and the analysis of the DnaA binding site in the promoter region (Polaczek and Wright 1990), we have suggested that these sequences, as part of an ancestral origin of replication (Yoshikawa and Ogasawara 1991), may have only limited or no regulatory functions (Polaczek 1998). IHF is a sequence-speci®c bending protein that relies heavily on structural recognition in addition to speci®c base contacts (Rice et al. 1996). The introduction of a GATC motif into the nonconserved spacer region of the oriC IHF site positions two GATCs within one turn of the helix, creating a site identical to the one present at the dnaA locus (Fig. 3). We suggest that this structural alteration results in the reduced anity of IHF for the site. This does not rule out the possibility that the ¯anking sequence of dnaA may further destabilize the complex nor does it rule out that other base substi-
tutions within the nonconserved spacer region may enhance or reduce the anity of IHF for the modi®ed site. What is interesting is that the 4-bp nonconserved region of the IHF binding site may be important for IHF binding. GATC sites are concentrated at E. coli oriC in regions of IHF protein contact (Polaczek 1990; Schaechter et al. 1991; Polaczek et al. 1997) and in the 13-bp repeats that compose the site of DNA unwinding (Bramhill and Kornberg 1988), suggesting a role for GATCs in protein recognition as well as helix destabilization. Our attempts to demonstrate an eect of GATCs and methylation on 13mer unwinding were inconclusive, possibly due to an overriding contribution of DNA supercoiling to the reaction (Polaczek et al. 1998). Results presented here show only subtle eects of GATCs in dierent states of methylation on electrophoretic migration of selected polylinkers, and these appear to be sequence context dependent. Nevertheless, methylation of GATCs significantly alters the IHF-induced bend at oriC (Polaczek et al. 1997) and has profound eects on intrinsically curved sequences adjacent to oriC (Kimura et al. 1989). Based on the results presented here it is reasonable to assume that only certain GATCs, in speci®c sequence contexts and properly phased with the larger bends, may contribute to the overall geometry of the oriC template. Evidence pointing to a physiological signi®cance of these structural alterations is indirect but, in our view, compelling. For example, SeqA, a negative regulator of DNA replication, binds to sequences containing hemimethylated GATCs but can also speci®cally bind to a unique fully methylated site (Slater et al. 1995). Apparently, in the particular sequence context, this site is structurally distinct from other GATCs. The methylation status of three GATCs also appears to control binding of Lrp regulatory protein to the T-rich site in the fae operon and aect the stability of the DNA-protein complexes formed (Huisman and de Graaf 1995). Finally, GATCs within the oriC IHF recognition sequence
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have modulatory, methylation-dependent, eects on IHF binding (Polaczek et al. 1997). In conclusion, GATC motifs are important regulatory elements in many cellular functions. The local structural alterations imposed by GATC motifs in different neighboring sequences and/or reciprocal actions of both elements lead to alterations in DNA conformation which may contribute to direct protein recognition of the GATCs, as well as having modulatory properties in indirect interactions. Acknowledgements We thank Howard Nash (National Institutes of Health) for the gift of IHF protein. This work was carried out with the support of National Institutes of Health grant GM25508
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