J. Mol. Biol. (1995) 252, 70–85
DNA Palindromes Adopt a Methylation-resistant Conformation that is Consistent with DNA Cruciform or Hairpin Formation in Vivo Thorsten Allers and David R. F. Leach* Institute of Cell and Molecular Biology University of Edinburgh Kings Buildings Edinburgh E9 3JR, UK
Long DNA palindromes present a threat to genomic stability and are not tolerated in Escherichia coli. It has been suggested that this is a consequence of cruciform or hairpin formation by palindromic sequences. This work describes a methylation inhibition assay for unusual DNA secondary structure in vivo that is both internally controlled and non-invasive. If a palindrome with a central GATC target site for Dam methylase assumes a cruciform or hairpin conformation in vivo, then the GATC sequence will be located in a single-stranded loop and will consequently not be modified. The centre of a long perfect palindrome located in bacteriophage l is shown to be methylation-resistant in vivo. Changes to the central sequence and insertions of 10 base-pairs of asymmetric sequence do not alter the degree of under-methylation, but insertions of 20 base-pairs or more of asymmetric sequence reduce the under-methylation of the palindrome centre. We also show that the centres of long palindromes are more under-methylated than equivalent sequences in a non-palindromic context. These results are consistent with an unusual secondary structure, such as DNA cruciform or hairpin, and indicate that the formation pathway of the structure detected is independent of the composition and symmetry of the central 10 base-pairs of the palindrome. 7 1995 Academic Press Limited
Keywords: DNA palindrome; DNA structure; cruciform DNA; Escherichia coli; Dam methylase
Introduction Long perfect DNA palindromes cannot be propagated in wild-type Escherichia coli (Collins, 1981; Lilley, 1981; Collins et al., 1982; Mizuuchi et al., 1982; Leach & Stahl, 1983), and vectors containing such sequences suffer two fates: the palindrome is either partially or completely deleted (instability), or proves lethal to the vector, which is not recovered (inviability). Inviability is associated with the inhibition of DNA replication (Leach & Lindsey, 1986; Shurvinton et al., 1987; Lindsey & Leach, 1989). Instability is almost certainly the result of replication slippage on the lagging strand of the replication fork (Trinh & Sinden, 1991), and is mediated by short direct repeats flanking the palindrome (Glickman & Ripley, 1984). Inviability is only encountered with palindromes greater than 150 to 200 base-pairs (bp) total length (Warren & Green, 1985), while instability has been detected with palindromes as short as 22 bp
Abbreviations used: bp, base-pair(s). 0022–2836/95/360070–16 $12.00/0
(DasGupta et al., 1987). Palindrome instability has also been observed in other organisms, including Saccharomyces cerevisiae (Gordenin et al., 1993; Henderson & Petes, 1993; Ruskin & Fink, 1993), and palindromic sequences in heteroduplex DNA have been found to avoid mismatch repair in S. cerevisiae (Nag et al., 1989). It has been proposed that these phenomena are facilitated by the formation of cruciform or hairpin structures from palindromic DNA (reviewed by Leach, 1994). The detection and quantification of cruciform DNA in vivo is problematic, not least because of the inviability and instability associated with long DNA palindromes. Inviability is largely overcome in E. coli sbcC or sbcD mutant hosts (Chalker et al., 1988; Gibson et al., 1992), and these mutants have therefore permitted the study of long DNA palindromes in vivo. In such hosts, the plaque size of l phage carrying long palindromes was found to be determined by the central sequence of the palindrome (Davison & Leach, 1994a,b) and the presence of small non-palindromic insertions at the centre of symmetry (Chalker et al., 1993); these findings argue 7 1995 Academic Press Limited
Methylation Assay for Cruciform or Hairpin DNA
for hairpin or cruciform formation in vivo. However, the direct examination of cruciform DNA in vivo remains difficult, as structural artifacts are easily introduced during DNA purification (Hyrien, 1989). Successful studies have therefore used in situ chemical probes (reviewed by Palecek, 1992; Sinden & Ussery, 1992) or cruciform-specific antibodies (Frappier et al., 1987), and several accounts of cruciform extrusion in E. coli have been published (Panayotatos & Fontaine, 1987; McClellan et al., 1990; Zheng et al., 1991). However, these workers used palindromes consisting of (A + T)-rich sequences, or palindromes flanked by (A + T)-rich DNA; cruciform extrusion by such sequences in vitro proceeds via unusual pathways (Greaves et al., 1985; Lilley, 1985). In fact, in vitro studies have suggested that cruciform extrusion by palindromes of a balanced base composition may be kinetically forbidden under physiological conditions (Courey & Wang, 1983; Gellert et al., 1983). Investigations using such palindromes have, until now, provided little direct evidence of cruciform extrusion in E. coli (Sinden et al., 1983). DNA methyltransferases have recently emerged as useful probes of DNA structure in vivo (Zacharias, 1992), as the method is non-invasive and measures the steady-state concentrations of methylation-sensitive and resistant structures. The inhibition of methylation at target sites that are in regions of unusual DNA secondary structure or are obscured by protein binding can be detected after DNA purification by cleavage with methylation-sensitive restriction endonucleases. Unlike the approaches outlined above, this method does not remove the products of DNA structural transitions, and will therefore not affect the equilibrium of a process such as cruciform extrusion. Dam methylase, which is endogenous to E. coli, modifies the adenine residue of GATC sequences, and both methylated and unmethylated targets can be independently quantified by restriction analysis. It has been used to investigate triplex H-DNA formation (Parniewski et al., 1990) and protein–DNA interactions (Wang & Church, 1992) in E. coli, and chromatin structure in yeast (Singh & Klar, 1992). In the internally controlled assay developed here, Dam methylase is used to probe for the presence of a single-stranded GATC loop at the palindrome centre, which may result from cruciform or hairpin formation in vivo. The ability of cruciform loops to inhibit DNA methylation has been established in vitro (Murchie & Lilley, 1989). Bacteriophage l is used as a vector because it is methylated to approximately 65% by Dam methylase in vivo, providing a sensitive substrate for the detection of methylation inhibition. We show here that a GATC site at the centre of a long DNA palindrome is under-methylated in vivo, and that central base substitutions and insertions of 10 bp of asymmetric sequence do not affect the degree of under-methylation; longer asymmetric insertions reduce the methylation-resistance of the palindrome centre. We also show that the length of the DNA palindrome
affects the under-methylation of its centre. These results indicate that palindromes adopt a methylation-resistant structure in E. coli, and are consistent with the formation of hairpin or cruciform structures by a pathway involving single-stranded palindromic DNA.
Results The centres of long perfect DNA palindromes are under-methylated in vivo The symmetrical oligonucleotides shown in Table 1 were inserted at the central SacI site of a 462 bp perfect palindrome in bacteriophage lDRL167. The palindromes therefore contained a central GATC target site which would be inaccessible to Dam methylase when the DNA was in a cruciform or hairpin conformation. These oligonucleotide insertions have been verified by the subcloning and sequencing of the palindrome centre. Oligonucleotides with concerted changes to bases immediately outside the GATC site were chosen because the sequence composition of a palindrome centre has been shown to influence both the rate of cruciform extrusion in vitro (Murchie & Lilley, 1987; Courey & Wang, 1988; Zheng & Sinden, 1988) and the plating of l phage bearing a long perfect palindrome on an sbcC host (Davison & Leach, 1994a,b). The phage were methylated by growth in sbcC (N2364) or recBC sbcBC (JC7623 or JC9387) hosts, which are permissive for long DNA palindromes. The l DNA was then analysed for Dam methylation using the methylation-sensitive restriction endonucleases DpnI (Lacks & Greenberg, 1975) and MboI (Gelinas et al., 1977), which cleave fully methylated and fully unmethylated GATC sequences, respectively. However, there are 116 GATC targets in (wild-type) l DNA, and many more DNA fragments than this will be formed by what is essentially a methylation-dependent partial restriction digest. In order to simplify the analysis of Dam methylation, the l DNA was initially cut to completion with EcoRI and radiolabelled at these EcoRI sites using Table 1. Bacteriophage l strains constructed by insertion of the oligonucleotides shown in the SacI site at the palindrome centre of lDRL167
The sequences shown in bold comprise target sites for the restriction endonucleases shown; all contain central GATC sequences.
Methylation Assay for Cruciform or Hairpin DNA
Figure 1. Map of lDRL176–179 (not to scale) showing the (a-35S)-labelled EcoRI sites and the GATC sites assayed for methylation (numbers indicate position in wild-type l sequence; Sanger et al., 1982). In lDRL167 the EcoRI B fragment of l is replaced by a 462 bp perfect palindrome (double arrow) with a central SacI site (see also Figure 7).
[a-35S]dATP and Klenow enzyme. If quantification is confined to DNA fragments with radiolabelled EcoRI termini, then the measurement of Dam methylation is greatly simplified. Cleavage by DpnI or MboI at GATC sites that are immediately adjacent to the radiolabelled EcoRI sites produces a discrete number of DNA fragments of predictable sizes, which can be quantified after gel electrophoresis; the palindromes in lDRL176–179 are flanked symmetrically by EcoRI sites and have only one (central) GATC site. The intensity of these EcoRI–GATC fragments will be directly proportional to the efficiency of DpnI or MboI cleavage at that GATC site. The position of these GATC targets and the sizes of predicted fragments are illustrated in Figure 1. The GATC sites lying outside the palindrome represent control targets for Dam methylase, as they are not expected to adopt a methylation-resistant DNA structure in vivo. The methylation of the palindrome centre can be compared to these other GATC sites, thereby ensuring that the experiment is internally controlled. Such a methylation-sensitive restriction digest is presented in Figure 2. In order to compare the efficiency of cleavage by DpnI or MboI at the GATC sites, an internal standard must be established. We have chosen to use the average of the quantified values for the control bands as a standard, as this will represent the typical level of Dam methylation outside the palindrome as detected by DpnI or MboI digestion. Thus a comparison of bands, such as those shown in Figure 2, with the average of the control bands, will indicate the relative efficiency of cleavage by DpnI or MboI. Such a comparison is shown in Figure 3 for lDRL177. Figure 3 also presents the data resulting from in vitro methylation of unmethylated lDRL177 DNA with Dam methylase. Target sites for DNA restriction and modification enzymes differ in their efficiency of cleavage or modification. It is therefore important to consider whether under-methylation observed at the palindrome centre is the result of methylation inhibition by a cruciform or hairpin structure, or merely due to the sequence context of that GATC site. Unmodified lDRL176–179 DNA was therefore prepared by growth in a recBC sbcBC dam (JL32) host and methylated in vitro using purified Dam methylase. The in vitro methylation is a control for any effect of primary DNA sequence context on methylation, since cruciform or hairpin formation is energetically unfavourable in linear relaxed DNA.
The in vitro-modified l DNA was analysed for Dam methylation at GATC sites as described above. l DNA which had been methylated in vitro to a similar
Figure 2. DpnI and MboI restriction digests of bacteriophage lDRL176–179 DNA, determining the level of Dam methylation at various GATC sites in vivo. l DNA was purified, cut with EcoRI, radiolabelled with [a-35S]dATP and Klenow enzyme, and cut with either DpnI or MboI as described in Materials and Methods. Bands are labelled that represent digestion at the GATC site in the palindrome centre or at control GATC sites outside the palindrome (see Figure 1). DpnI cuts only fully methylated GATC and MboI cuts only unmethylated GATC.
Methylation Assay for Cruciform or Hairpin DNA
Figure 3. Relative efficiency of cleavage by DpnI (a) and MboI (b) at GATC sites in lDRL177 following methylation in vivo ( ) in N2364 (sbcC) or in vitro (Q) using purified Dam methylase. Band intensities are presented relative to the average intensity of the control bands; the value for the half palindrome band was halved to correct for radiolabelling at both EcoRI sites (see Figure 1). DpnI cleaves only methylated GATC, whereas MboI cleaves only unmethylated GATC.
extent to the DNA prepared on dam+ hosts was chosen for analysis. The side-by-side comparison of in vivo and in vitro methylation efficiencies presented in Figure 3 shows; 1. After in vitro methylation, the control GATC sites display relative efficiencies of
DpnI and MboI cleavage that are similar to those observed after methylation in vivo; 2. The palindrome centre of lDRL177 is cleaved with a lower relative efficiency by DpnI after in vivo methylation than after in vitro methylation, and, conversely, is
74 cleaved with a higher relative efficiency by MboI after in vivo methylation than after in vitro methylation. As DpnI cleaves only at methylated GATC and MboI cleaves only at unmethylated GATC, these results show that the palindrome centre of lDRL177, but not any of the control GATC sites, is relatively under-methylated in vivo. As the GATC site at the palindrome centre was inserted as an unmethylated oligonucleotide, this target will be relatively under-methylated for the first rounds of DNA replication. However, unlike Type I restriction/modification DNA methyltransferases, Dam methylase does not show a significant kinetic preference for hemimethylated, as opposed to an unmethylated, target sites (Herman & Modrich, 1982); pre-existing differences between the oligonucleotide insertion and other GATC sites should therefore be obscured by successive rounds of replication and methylation. In order to verify that this initial under-methylation is not persistent, a l derivative with such an oligonucleotide insertion was passaged through isogenic dam− (JL32) or dam+ (JC9387) strains (which were otherwise recBC sbcBC), and thereafter grown on a dam+ sbcC (N2364) host. The passage through the dam− host should demethylate the l DNA, and thereby remove any bias due to pre-existing under-methylation. Methylated DNA was prepared from both l strains and analysed for Dam methylation; no significant difference was found at any of the GATC sites examined (data not shown).
Methylation Assay for Cruciform or Hairpin DNA
The in vivo and in vitro methylation efficiencies of lDRL176, lDRL178 and lDRL179 were compared and are presented in Figure 4, which shows the relative cleavage by DpnI at the palindrome centre GATC site. In each case, the control GATC sites displayed similar relative efficiencies of methylation in vivo and in vitro (data not shown; see Figure 3 for lDRL177 data). Figure 4 shows that the palindrome centres of lDRL176–179 are all relatively undermethylated in vivo; this significant under-methylation was not found at any of the control sites (data not shown; see Figure 3 for lDRL177 data). Although the relative levels of in vivo methylation at the palindrome centres appear to differ between the phage strains (and therefore between the palindrome central sequences), these differences are cancelled out by the methylation efficiencies measured in vitro. This is best illustrated by Figure 4: for example, compared to lDRL179, the palindrome centre in lDRL176 is cleaved relatively efficiently by DpnI after both in vivo and in vitro methylation. However, in all cases, the DpnI cleavage after in vivo methylation is only 0.35 to 0.41 times as efficient as after in vitro methylation. This indicates that the base sequence of the central 6 bp of a palindrome does not significantly affect the degree of its under-methylation in vivo, and by implication, does not influence the formation and/or persistence of a methylation-resistant structure at that site. Similar data were obtained for phage prepared on a recBC sbcBC (JC7623 or JC9387) host (data not shown).
Figure 4. Relative efficiency of cleavage by DpnI at the palindrome centre GATC site in lDRL176–179 following methylation in vivo ( ) in N2364 (sbcC) or in vitro (Q) using purified Dam methylase. Levels of cleavage are relative to the average of the control GATC sites (see Figure 3).
Methylation Assay for Cruciform or Hairpin DNA
Table 2. Bacteriophage l strains constructed by insertion of the asymmetric oligonucleotides shown in the SacI site at the palindrome centre of lDRL167
site in vivo. The palindrome centre of lDRL180 has a balanced base composition (50% G/C), whereas the palindrome centres of lDRL188 and lDRL189 are A/T-rich (30% G/C) and G/C-rich (70% G/C), respectively. These oligonucleotide insertions have been verified by the subcloning and sequencing of the palindrome centre. lDRL180, lDRL188 and lDRL189 were methylated by growth in sbcC (N2364), recBC sbcBC (JC9387) or recA recD sbcC (N2694) hosts, and analysed for Dam methylation using DpnI or MboI as described previously. Alternatively, unmethylated DNA was generated by growth in a recBC sbcBC dam (JL32) host, and methylated in vitro using Dam methylase; this in vitro-modified l DNA was also analysed for Dam methylation. The comparison of in vivo and in vitro methylation efficiencies presented in Figure 5 shows that the palindrome centres of lDRL180, lDRL188 and lDRL189 are under-methylated in vivo; this under-methylation is not found at the control sites (data not shown). DpnI cleavage at the palindrome centres of lDRL180, lDRL188 and lDRL189 after in vivo methylation is only 0.35 to 0.41 times as efficient as after in vitro methylation. These values are very similar to those obtained for the perfect palindromes in lDRL176–179 (0.35 to 0.41). This implies that the insertion of 10 bp of asymmetry at the centre of a palindrome does not affect the formation and/or persistence of the methylationresistant structure in vivo. Furthermore, the similarity between the levels of under-methylation seen at the palindrome GATC sites of lDRL180, lDRL188 and lDRL189 indicates that the base composition of an asymmetric palindrome centre does not affect the formation and/or persistence of
The centres of long DNA palindromes with 10 bp of asymmetry are under-methylated in vivo The effect of small asymmetric central insertions on the under-methylation of the palindrome centre in vivo was examined. The non-palindromic oligonucleotides shown in Table 2 were inserted at the central SacI site of the 462 bp perfect palindrome in lDRL167, as described previously. The oligonucleotides in Table 2 form the complementary pairs shown, and with the exception of the central GATC site, have no inverted symmetry over a 10 bp interval. As the central bases cannot be expected to contribute to hairpin or cruciform formation on their own, the oligonucleotide pairs form an effective asymmetry of 10 bp at the palindrome centre. Asymmetric oligonucleotides differing in their base composition were chosen, in order to determine whether the central sequence of an interrupted palindrome affects the efficiency of methylation at a central GATC
Figure 5. Relative efficiency of cleavage by DpnI at the palindrome centre GATC site in lDRL180, lDRL188 and lDRL189 following methylation in vivo ( ) in N2364 (sbcC) or in vitro (Q) using purified Dam methylase. Levels of cleavage are relative to the average of the control GATC sites (see legend to Figure 3).
Methylation Assay for Cruciform or Hairpin DNA Table 3. Bacteriophage l strains constructed by insertion of the asymmetric oligonucleotides shown in the SacI site at the palindrome centre of lDRL167
the methylation-resistant structure in vivo either. Similar data were obtained for phage prepared on recBC sbcBC (JC9387) or recA recD sbcC (N2694) hosts (data not shown). The centres of long DNA palindromes with 20, 75 and 154 bp of asymmetry show reduced under-methylation in vivo In order to investigate further the effect of asymmetry at the palindrome centre on methylation of a central GATC site, the oligonucleotides shown in Table 3 were inserted into the SacI site at the centre of the 462 bp palindrome in lDRL167. These oligonucleotides form asymmetric insertions with a GATC site at their centre, and the sequence between this central target and the SacI-compatible end is essentially random. The central 10 bp of the oligonucleotide insertion in lDRL216 (20 bp asymmetry) are identical to the central 10 bp of lDRL180 (10 bp asymmetry), and the central 20 bp of the oligonucleotide insertion in lDRL218 (75 bp asymmetry) are identical to the central 20 bp of lDRL216 (20 bp asymmetry). An asymmetric sequence of
75 bp was chosen for the construction of lDRL218, as this is longer than the 57 bp previously determined to be the minimum required for an alleviation of inviability in a plasmid system (Warren & Green, 1985). However, it was found that, in spite of this 75 bp interruption, lDRL218 was still unable to plate efficiently on an sbcC+ (594) host. In order to determine the minimum length of asymmetry required for the complete alleviation of l inviability, the 75 bp asymmetric oligonucleotide was phosphorylated to promote the insertion of multimers, and potential l clones were selected by plating on an sbcC+ (594) host. An insertion of two oligonucleotides, or 154 bp, was found to be necessary to permit growth on the sbcC+ host; this l clone was designated lDRL219. lDRL216, lDRL218 and lDRL219 were methylated by growth in an sbcC (N2364) host and analysed for Dam methylation using DpnI as described previously. Alternatively, unmethylated DNA was generated by growth in a recBC sbcBC dam (JL32) host, methylated in vitro using Dam methylase, and analysed for Dam methylation. Both sets of data are presented in Figure 6. This comparison of in vivo and in vitro methylation efficiencies shows that, while the
Figure 6. Relative efficiency of cleavage by DpnI at the palindrome centre GATC site in lDRL216, lDRL218 and lDRL219 following methylation in vivo ( ) in N2364 (sbcC) or in vitro (Q) using purified Dam methylase. Levels of cleavage are relative to the average of the control GATC sites (see legend to Figure 3).
Methylation Assay for Cruciform or Hairpin DNA Table 4. Bacteriophage l strains constructed by insertion of the asymmetric oligonucleotides shown in the SacI site of lDRL152
Palindrome types and analogous bacteriophage strains constructed by insertion of identical oligonucleotides at the palindrome centre of lDRL167 are shown.
palindrome centres of lDRL216, lDRL218 and lDRL219 are under-methylated in vivo, the difference between the efficiency of methylation in vivo and in vitro is not as large as in the cases of lDRL176–180 and lDRL188–189: DpnI cleavage at the palindrome centres of lDRL216, lDRL218 and lDRL219 after in vivo methylation is 0.56 to 0.63 times as efficient as after in vitro methylation. These values indicate less under-methylation than at the centre of the perfect palindromes in lDRL176–179 (0.35 to 0.41) and the palindromes with 10 bp of asymmetry in lDRL180, lDRL188 and lDRL189 (0.35 to 0.41). This implies that asymmetric insertions of 20 bp or more reduce, but do not abolish, the formation and/or persistence in vivo of the methylation resistant structure at the palindrome centre. Furthermore, the similarity between the values for lDRL216, lDRL218 and lDRL219
indicates that asymmetric insertions of 75 or 154 bp reduce the formation and/or persistence of this structure no more than insertions of 20 bp of asymmetric sequence. The centres of long DNA palindromes are more under-methylated in vivo than equivalent sequences in a non-palindromic context In order to determine the contribution of palindrome length to the under-methylation of GATC sites at its centre, the oligonucleotides shown in Table 4 were inserted into the unique SacI site of lDRL152. This phage is isogenic to lDRL167, except that in the latter the EcoRI B fragment of l (21226 bp to 26104 bp of the wild-type l sequence; Sanger et al., 1982) is replaced by an inverted duplication of the SacI-EcoRI fragment (25881 bp to 26104 bp). This is
Figure 7. Maps of lDRL152 and lDRL167 (not to scale) illustrating the replacement of the EcoRI B fragment of lDRL152 (21226 bp to 26104 bp) with an inverted duplication of the SacI-EcoRI fragment (25881 bp to 26104 bp) to generate the 462 bp perfect palindrome (double arrow) in lDRL167 (numbers indicate position in wild-type l sequence; Sanger et al., 1982).
Methylation Assay for Cruciform or Hairpin DNA
Figure 8. Relative efficiency of cleavage by DpnI at the oligonucleotide insert GATC site in derivatives of lDRL152 (which has no long palindrome; lDRL192–195, lDRL212–215 and lDRL217; ) and derivatives of lDRL167 (which has a 462 bp palindrome; lDRL176–180, lDRL188–189, lDRL216 and lDRL218; Q) following methylation in vivo in N2364 (sbcC). Levels of cleavage are relative to the average of the control GATC sites (see legend to Figure 3). Numbers in parentheses on the abscissa refer to lengths of central asymmetry (where present).
illustrated in Figure 7. The perfectly symmetrical oligonucleotides which have previously been cloned in lDRL176–179 will therefore form small perfect palindromes of 20 bp in lDRL212, lDRL213, lDRL192 and lDLR193, respectively. The oligonucleotides with 10 bp interruptions which have previously been cloned in lDRL180, lDRL188 and lDRL189 will form small inverted repeats of five bp, separated by 10 bp of asymmetry, in lDRL214, lDRL195 and lDRL194, respectively. Similarly, the oligonucleotides with 20 and 75 bp interruptions which have previously been cloned in lDRL216 and lDRL218 will form small inverted repeats of five bp, separated by 20 and 75 bp of asymmetry, in lDRL215 and lDRL217, respectively. lDRL192–195, lDRL212–215 and lDRL217, were methylated by growth in an sbcC (N2364) host, and analysed for Dam methylation using DpnI as described previously. The data are presented in Figure 8, in conjunction with data described previously for l strains constructed by insertion of the same oligonucleotides at the palindrome centre of lDRL167. Figure 8 therefore shows the effect of palindrome length on the methylation in vivo of a central GATC site. The data show that after in vivo methylation, each oligonucleotide insertion is
cleaved more efficiently by DpnI when cloned in lDRL152 than when cloned in lDRL167. This indicates that GATC sites in oligonucleotide insertions are methylated more efficiently in lDRL152 than in lDRL167. In other words, the insertions are more under-methylated at the centre of a long palindrome than in a non-palindromic context. lDRL217 was also chosen as a control to determine whether the region around the SacI site, which contains the long palindrome in lDRL167 derivatives, is under-methylated in vivo even if no unusual DNA secondary structure is formed. lDRL217 is a palindrome-free derivative of lDRL152, and has an insertion of 75 bp of non-palindromic sequence with a central GATC site; an insert of this size was used to separate the five bp inverted repeats of the inactivated SacI target site, which could otherwise form a small hairpin in vivo. Unmethylated lDRL217 DNA was also prepared by growth in a recBC sbcBC dam (JL32) host, methylated in vitro using Dam methylase, and analysed for Dam methylation. The efficiency of in vivo methylation at the GATC site in the oligonucleotide insertion present in lDRL217 did not differ significantly from that observed in vitro (data not shown), confirming that this region of bacteriophage l is methylated normally in vivo.
Methylation Assay for Cruciform or Hairpin DNA
Discussion The centres of long perfect palindromes are under-methylated in vivo We show that the centres of long DNA palindromes in a bacteriophage l derivative adopt a methylation-resistant conformation when grown in an E. coli sbcC strain. Similar results have been reported elsewhere: Dam methylation-resistant sites in plasmids were found in inverted repeats, and have been linked to the control of plasmid replication (Russell & Zinder, 1987). The work presented here extends this finding by investigating the effect of palindrome length, symmetry and central sequence composition on the formation of a methylation-resistant structure. Furthermore, the palindromes used in this work are not located near the origin of replication and do not form part of any known operator sequence, and should therefore be free of extensive protein–DNA interactions that may prevent accessibility to methylases. The GATC sites analysed in l differ in their efficiency of Dam methylation, as illustrated in Figure 3. This is partly due to the target site preference exhibited by Dam methylase, which has been shown to differ in its affinity for GATC sites located in different sequence contexts (Bergerat et al., 1989). In order to take this site preference into account, unmethylated l DNA was prepared and methylated in vitro under conditions which make the formation of unusual DNA secondary structures, which would inhibit methylation, unfavourable. A similar pattern of relative methylation efficiency was found after both in vivo and in vitro methylation at all the control GATC sites, but not in the case of the target located at the centre of the long perfect palindrome; this GATC site shows significant under-methylation in vivo. We show in Figure 4 that sequence changes at the centre of a long perfect palindrome affect the efficiency of Dam methylation both in vivo and
79 in vitro, but in each case the palindrome centre is significantly under-methylated in vivo. In fact, the ratio of in vivo to in vitro methylation efficiency is remarkably resistant to base sequence changes. This indicates that if the under-methylation is the consequence of a methylation-resistant structure, then the formation and/or persistence of this structure is unaffected by sequence alterations at the palindrome centre. This argues against its formation by a pathway such as spontaneous cruciform extrusion, which is dependent on central sequence composition; in vitro studies have shown that cruciform extrusion is initiated by melting of the central 10 bp of the palindrome (Murchie & Lilley, 1987; Courey & Wang, 1988; Zheng & Sinden, 1988), and is therefore affected by the thermal stability of this sequence. However, if cruciform formation in vivo were initiated by assisted unwinding of the palindrome centre (for example, during transcription or DNA replication), then the formation of the methylationresistant structure would not be affected by the composition of the central sequence. Alternatively, formation of a DNA hairpin from single-stranded palindromic DNA relies only on inverted symmetry and not on the central base sequence; such a process would also account for the data presented here, and might similarly occur during transcription or DNA replication. The difference between centre-dependent and centre-independent cruciform extrusion and hairpin formation is illustrated in Figure 9. The centre-independence of methylation-inhibition by long palindromes contrasts with the results of Davison & Leach (1994a,b), who found that plaque size on sbcC mutant strains of l phage bearing long palindromes is affected by the composition of the palindrome centre, albeit in a different manner to cruciform extrusion in vitro. It was found that central sequence changes had a greater effect on plaque size than peripheral changes, and that plaque size could be correlated with the stability of base-pairing in potential DNA hairpins. These results argue that intrastrand pairing within palindromes is important
Figure 9. Schematic diagram illustrating (a) Cruciform extrusion by a double-stranded DNA palindrome via spontaneous central sequence melting and negative supercoilingdriven branch migration (Murchie & Lilley, 1987; Courey & Wang, 1988; Zheng & Sinden, 1988). (b) Cruciform extrusion initiated by assisted DNA unwinding. (c) Hairpin formation by a single-stranded DNA palindrome.
80 in determining their effect in vivo, and therefore suggest the involvement of a cruciform or hairpin structure. Similar measurements of l plaque size have been undertaken using lDRL176–180 (data not shown), and the results concur with the findings of Davison & Leach (1994a,b). The disparity between the effects of central sequences in these two assays indicates that they detect different structures, or measure different aspects or processing of the same structure. The first possibility could arise if the methylation assay detects hairpins that are formed by a centre-independent pathway, whereas the plaque assay is sensitive to cruciforms that are formed by a centre-dependent process. On the other hand, both assays may detect the same structure, but lead to different conclusions if they are sensitive to different physical or temporal aspects of the structure. For example, if cruciform or hairpin formation is centreindependent, the methylation assay will detect the presence of the structure per se and will reflect this centre-independence, while the plaque size assay may be sensitive to the problems incurred during the replication of DNA hairpins; this could account for the relationship between plaque size and the theoretical stability of intrastrand pairing in such hairpins (Davison & Leach, 1994a,b). Alternatively, both assays may measure the formation of cruciforms and/or hairpins, but at different times in the l life-cycle; the plaque assay may be sensitive to centre-dependent structure formation in early (u) replication, whereas the methylation assay could detect a centre-independent reaction during late (rolling-circle) replication. In this case, the assay presented here may fail to detect the centre-dependent formation of methylation-resistant structures by palindromes that are replicated poorly in the u phase and consequently lead to small plaques. However, this would imply that host mutations in recBCD may affect the methylation assay. To proceed from u to rolling-circle replication, l derivatives require either a functional gam gene product to inactivate the host RecBCD exonuclease (Enquist & Skalka, 1973), or the presence of a x recombination hotspot; x both promotes the formation of viable l dimers by RecBCD-mediated homologous recombination (Smith, 1988) and protects the double-stranded DNA tails produced during rolling-circle replication from RecBCD-mediated degradation (Dabert et al., 1992). The l derivatives used here are red− gam− x+, and were grown in both E. coli sbcC and recBC sbcBC hosts; the latter permits efficient rolling-circle replication of both x+ and x-free phage. The in vivo methylation efficiencies did not differ significantly, suggesting that the formation of methylation-resistant structures is not affected by an increase in rolling-circle replication or by any concomitant changes in DNA topology. The resistance of under-methylation to base sequence changes argues against methylation-inhibition by protein binding. The target sites of protein–DNA interaction are frequently palindromic, and certain GATC sites on the E. coli chromosome have been found to be methylated
Methylation Assay for Cruciform or Hairpin DNA
slowly, due to the sequestration of that region by cellular enzymes (Campbell & Kleckner, 1990). However, as all the perfect palindrome centres studied display similar levels of under-methylation in spite of central sequence alterations, it is unlikely that the accidental formation of a protein binding site is responsible for the methylation inhibition. An alternative form of protein–DNA interaction that might account for under-methylation is the RecAmediated pairing of palindrome arms. The two arms of the palindrome are homologous, and RecA could theoretically pair these duplexes to form a nucleoprotein filament incorporating four DNA strands (Conley & West, 1989). The GATC site at the centre of the palindrome would then be inaccessible to Dam methylase. However, in vivo methylation of the palindrome centre of l phage grown in the recA recD sbcC host did not differ significantly from that observed in phage prepared on an sbcC host, confirming that RecA-mediated pairing is not responsible for the under-methylation.
Effects of asymmetry at the centre of a long palindrome on its under-methylation Central interruptions to the inverted symmetry of a long palindrome can overcome the inviability associated with such sequences (Collins et al., 1982; Mizuuchi et al., 1982); the minimum length of asymmetry required for the complete alleviation of inviability in wild-type E. coli has been determined to be 60 to 150 bp (Warren & Green, 1985). However, smaller lengths of central asymmetry may have intermediate effects: small asymmetric insertions up to 27 bp in length partially alleviate the residual inviability of l phage bearing a long perfect palindrome in several mutant hosts (Chalker et al., 1993), and the severity of inviability declines with an increasing length of asymmetry. The effect of asymmetric insertions on under-methylation of the palindrome centre in vivo was therefore examined. We show that neither the presence nor composition of 10 bp of asymmetric sequence (including a GATC site) at the centre of a long palindrome affects the level of under-methylation relative to that observed with perfect palindromes (Figure 5). This implies that the formation and/or persistence of the methylation-resistant structure incorporating the palindrome centre is unaffected by the insertion (and composition) of 10 bp of non-palindromic sequence. This rules out a centre-dependent pathway of structure formation, as melting at the palindrome centre would have to be extended by up to 10 bp before cruciform extrusion via the pathway shown in Figure 9(a) could be initiated; this has also been suggested by theoretical calculations (Hsieh & Wang, 1975; Warren & Green, 1985). On the other hand, if the structure responsible for under-methylation in vivo is formed by a pathway involving DNA unwinding, then the data presented here imply that the single-stranded region is longer than 10 bases. When 20, 75 or 154 bp of asymmetry are inserted
Methylation Assay for Cruciform or Hairpin DNA
at the palindrome centre, the methylation at a GATC site located therein is increased: relative levels of DpnI cleavage (which take into account the in vitro methylation data) are, on average, 1.55 times higher than those at the centres of the perfect palindromes and those with 10 bp of asymmetry. Relative levels of cleavage for all the derivatives of lDRL167 are presented in Figure 10. This increased methylation indicates that the insertion of 20 bp or more of asymmetry reduces the formation and/or persistence of the methylation-resistant structure incorporating the palindrome centre. However, the relative levels of DpnI cleavage do not differ significantly between the centres of palindromes with 20, 75 or 154 bp of asymmetry, indicating that insertions of 20 bp are as effective at reducing the formation and/or persistence of a structure as insertions of 154 bp. This may imply that the single-stranded window facilitating DNA cruciform or hairpin formation is less than 20 (but greater than 10) bases in effective length. However, as palindromes with 20 bp or more of asymmetry are still somewhat under-methylated, there must exist another pathway for the formation of a methylation-resistant structure
which is not sensitive to long asymmetric insertions. It is possible that both the centre-independent pathways illustrated in Figure 9 operate in vivo, but that in one of these the single-stranded region is between 10 and 20 bases in length, whereas in the other it is longer than 154 bases. A potential alternative explanation for these results is that the region of l containing the palindrome is under-methylated in vivo, even when no unusual DNA secondary structure is formed. However, when 75 bp of non-palindromic sequence were inserted in l at an equivalent position to the long palindrome, the in vivo methylation efficiency of a central GATC site was not found to differ significantly from that found in vitro. This indicates that the region containing the palindrome is methylated normally in vivo. It is noteworthy that only the l derivative with an asymmetric insertion of 154 bp was viable in a wild-type (sbcC+ ) E. coli host. This insert is significantly longer than the 57 bp of central asymmetry previously determined to be the minimum required for the alleviation of inviability (Warren & Green, 1985). However, the palindrome
Figure 10. Relative levels of DpnI cleavage at the palindrome centres of lDRL167 derivatives after methylation in vivo. The relative level of cleavage shown here compares the relative efficiency of DpnI cleavage after in vivo methylation to the relative efficiency of DpnI cleavage after in vitro methylation†; a relative cleavage of 100% would indicate no under-methylation. Numbers in parentheses on the abscissa refer to lengths of central asymmetry. † Relative DpnI cleavage here is calculated by:
PalindromeDpnI Average ControlDpnI
1 >0 In Vivo
PalindromeDpnI Average ControlDpnI
Methylation Assay for Cruciform or Hairpin DNA
used in that study was cloned in a plasmid vector, whereas the work described here uses a l derivative to propagate the palindrome. This difference may therefore be due to the modes of replication employed by plasmids and l phage, or the sensitivities of the assays used to determine inviability.
Effects of palindrome length on under-methylation at its centre The oligonucleotides which had been inserted at the centre of the long palindrome were also cloned in an isogenic l phage which does not have a long inverted repeat (Figure 7). A comparison of in vivo methylation at these GATC sites with the results obtained using l derivatives with long palindromes (Figure 8) shows that GATC sites are more methylation-resistant when at the centre of a long palindrome than when in a non-palindromic context. This implies that, regardless of the length of any asymmetry, the context of a long palindrome facilitates the formation and/or persistence of a methylation-resistant structure such as a DNA cruciform or hairpin.
Materials and Methods Unless otherwise indicated, enzymes were used according to the manufacturer’s instructions and manipulations were carried out according to standard methods (Sambrook et al., 1989).
Bacterial strains The E. coli K-12 strains used here are listed in Table 5. Apart from 594, all strains are derivatives of AB1157 (F− thi-1 his-4 D(gpt-proA)62 argE3 thr-1 leuB6 kdgK51 rfbD1 ara-14 lacY1 galK2 xyl-5 mtl-1 tsx-33 supE44 rpsL31; Howard-Flanders & Theriot, 1966).
Bacteriophage strains The bacteriophage strains used here are all derived from lDRL152 (l spi6 cI857 x+C) or lDRL167 (l DB pal spi6 cI857 x+C; Davison & Leach, 1994a), in which the EcoRI B fragment of l is replaced by a 462 bp perfect palindrome flanked by EcoRI sites. The oligonucleotides (Oswel DNA) shown in Tables 1 to 4 were inserted into the unique SacI site at the centre of the palindrome in lDRL167 (Figure 1)
or at the unique SacI site of lDRL152 (Figure 7). To facilitate the isolation and identification of desired clones, the oligonucleotides used here disrupted the SacI site and generally introduced a new restriction target sequence subsuming the GATC site (Tables 1 to 4). Potential clones were grown by plate lysis and l DNA was extracted using a small-scale method (described below). l DNA was analysed by digestion using the appropriate restriction endonucleases, followed by agarose or polyacrylamide gel electrophoresis. In the construction of lDRL217–219, it was found necessary to purify the complementary 79 base oligonucleotides by isolation from polyacrylamide gels, and to identify the desired clones by plaque blotting: l DNA was transferred to Hybond-N+ filters (Amersham) and probed using one of the 79 base oligonucleotides which had been radiolabelled using [g-32P]ATP and bacteriophage T4 polynucleotide kinase. Except in the case of lDRL219, the oligonucleotides were not phosphorylated to prevent the insertion of multimers; phosphorylation was carried out using T4 polynucleotide kinase. Growth of bacteriophage l in E. coli by plate lysis and small-scale extraction of l DNA An overnight culture of JC7623 or JC9387 was diluted 10-fold in L broth supplemented with 0.2% (w/v) maltose, 5 mM MgSO4 , 10 mg ml−1 vitamin B1 , and grown for 140 minutes at 37°C. An equal volume of 10 mM Tris-HCl (pH 7.5), 10 mM MgSO4 was added. A single l plaque was picked into 1 ml 10 mM Tris-HCl (pH 7.5), 10 mM MgSO4 and allowed to diffuse into solution for two hours. A 0.2 ml sample of the phage suspension was added to a 0.25 ml aliquot of the cell suspension, the phage were allowed to adsorb for 20 minutes at 37°C, and then poured onto fresh L agarose plates (L broth containing 12 g l−1 agarose, and supplemented with 10 mM TrisHCl (pH 7.5), 0.3% (w/v) glucose, 2 mM MgSO4 , 0.8 mM CaCl2 , 4 mM FeCl3 , 10 mg ml−1 vitamin B1 ) in 2.5 ml of molten L agarose top medium (L broth containing 4 g l−1 agarose). After six to eight hours incubation at 37°C, the top medium was homogenised with 6 ml 10 mM Tris-HCl (pH 7.5), 10 mM MgSO4 , 1% (v/v) chloroform, and l particles were allowed to diffuse overnight at 4°C; the suspension was cleared by centrifugation for 10 minutes at 3000 g and 4°C. A 1 ml sample of the suspension was incubated with 50 mg of DNase and 50 mg of RNase for 30 minutes at 37°C, then centrifuged briefly for one minute at 20,000 g. The supernatant was added to 0.5 ml ice-cold 30% (w/v) polyethylene glycol, 3 M NaCl and incubated on ice for four hours. After centrifugation for 15 minutes at 20,000 g and 4°C, the pellet was resuspended in 10 mM Tris-HCl (pH 7.5), 10 mM MgSO4 and extracted with chloroform, phenol, phenolchloroform (1:1 by vol.) and chloroform-isoamylalcohol (24:1, by vol.), and concentrated by two rounds of ethanol precipitation.
Table 5. E. coli K-12 strains used here Strain 594 JC7623 JC9387 JL32 N2364 N2694
F lac galK2 galT22 rpsL179 (Str ) recB21 recC22 sbcB15 sbcC201 recB21 recC22 sbcB15 sbcC201 sup o recB21 recC22 sbcB15 sbcC201 sup o dam3 (Strr ) xyl+ sbcC201 phoR79::Tn10 recA269::Tn10 recD1009 sbsC201 lac+
Reference Campbell (1965) Kushner et al. (1971) Bachmann (1987) Lindsey (1987) Lloyd & Buckman (1985) Chalker et al. (1988)
Methylation Assay for Cruciform or Hairpin DNA Growth of bacteriophage l in E. coli by liquid lysis and large-scale extraction of l DNA An overnight culture of E. coli was diluted 50-fold in L broth supplemented with 20 mM Tris-HCl (pH 7.5), 10 mM MgSO4 and grown, shaking at 37°C, to A650 = 0.5. The l phage were added at a multiplicity of infection of 0.1 and growth was continued, shaking at 37°C, for five to eight hours until complete lysis had occurred. Chloroform was added to a final concentration of 0.2% (v/v) and shaking continued for 10 minutes. The lysate was then centrifuged for 15 minutes at 17,500 g and 4°C to remove cell debris, incubated with 1 mg ml−1 of DNase and 1 mg ml−1 of RNase for 30 minutes at 37°C, and NaCl added to a final concentration of 1 M. After overnight incubation on ice, the lysate was cleared once more by centrifugation for 10 minutes at 17,500 g and 4°C, and polyethylene glycol was added to a final concentration of 10% (w/v). The lysate was incubated on ice for four hours and the phage harvested by centrifugation for 15 minutes at 25,000 g and 4°C. The pellet was resuspended in 6 ml 10 mM Tris-HCl (pH 7.5), 10 mM MgSO4 , extracted with chloroform and purified twice by CsCl step gradient centrifugation for 1 hour at 210,000 g and 18°C on a DuPont Sorvall TH641 rotor. The phage were collected by side-puncture using a hypodermic needle and dialysed against 10 mM Tris-HCl (pH 7.5), 1 mM EDTA overnight. l DNA was isolated by treatment with 1 mg ml−1 pronase during dialysis against 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.002% (v/v) Triton X-100, followed by phenol, phenolchloroform (1:1, by vol.) and chloroform-isoamylalcohol (24:1, by vol.) extraction, and concentrated by two rounds of ethanol precipitation.
In vitro methylation of l DNA Unmethylated l DNA was generated by growth in JL32 (recBC sbcBC dam). In vitro methylation was undertaken using Dam methylase (New England Biolabs) at a concentration of six units per mg l DNA in 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2 , 1 mM dithiothreitol, 80 mM S-adenosylmethionine for times ranging between 10 minutes and one hour at 37°C. The modified DNA was then purified by phenol-chloroform (1:1, by vol.) and chloroform-isoamylalcohol (24:1, by vol.) extraction and ethanol precipitation, and analysed for Dam methylation as described below. Analysis of Dam methylation l DNA was analysed for Dam methylation using DpnI and MboI (New England Biolabs), which cleave at methylated and unmethylated GATC targets respectively. A 2 mg sample of l DNA was digested to completion with 10 units EcoRI, and radiolabelled using 10 mCi [a-35S]dATP (0600 Ci mmol−1 ) and 2 units Klenow enzyme. The DNA was purified by phenol-chloroform (1:1, by vol.) and chloroform-isoamylalcohol (24:1, by vol.) extraction, and ethanol precipitation. The l DNA was divided into two samples and digested with 4 units of either DpnI or MboI. The DNA was purified by phenol-chloroform (1:1, by vol.) and chloroform-isoamylalcohol (24:1, by vol.) extraction and ethanol precipitation, and resuspended in formamideEDTA gel-loading sample buffer. Samples were denatured by boiling and electrophoresed on a 6% (w/v) polyacrylamide gel containing 7 M urea, which was run at 55°C to prevent hairpin formation by full-length DNA palindrome fragments. The gel was subsequently fixed in 10% (v/v) methanol, 10% (v/v) acetic acid, dried and
either autoradiographed or exposed to a storage phosphor screen. Quantification of bands was performed using a Molecular Dynamics Phosphorimager. In most cases only the DpnI cleavage data is presented. Subcloning and DNA sequencing of palindrome centres The palindrome centres of lDRL176–180 and lDRL188– 189 were subcloned and sequenced to confirm their base sequence; direct sequencing of the whole palindrome is impractical due to extensive secondary structure formation (Davison, 1994). After digestion with EcoRI, the palindromes were purified from agarose gels using a GeneClean kit (BIO 101). A 28 bp BstBI fragment containing the palindrome centre was then ligated into the AccI site of pMS2B, a derivative of pUC18 that stabilises long DNA palindromes (D. Leach, M. Shaw & C. Blake, unpublished results). Plasmid DNA was extracted using a QIAGEN plasmid midi kit (QIAGEN Inc.). Plasmids with inserts corresponding to the palindrome centre were identified by restriction analysis and sequenced using a Sequenase kit (U.S. Biochemical Corp.). Sequencing reactions were run on 6% (w/v) denaturing gradient polyacrylamide gels, which were fixed in 10% (v/v) methanol, 10% (v/v) acetic acid, dried and autoradiographed.
Acknowledgements We thank Catherine Blake, John Connelly and Angus Davison for many useful discussions and a critical reading of the manuscript, Ewa Okely for some preliminary experiments, and Michael Lichten for suggesting improvements to the manuscript. The work described here has been supported by a project grant from the Medical Research Council (to D.R.F.L.) and a studentship from the Science and Engineering Research Council (to T.A.).
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Methylation Assay for Cruciform or Hairpin DNA
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Edited by J. Karn (Received 18 April 1995; accepted 28 June 1995)