Nucleic Acids Research

INTRODUCTION

The site-specificity of classical post-replicative DNA modification in prokaryotes is determined by protein-DNA interactions involving recognition of cognate sequences typically 4-6 bp in length by a domain or subunit of the modifying enzyme (reviewed in 1). We have been investigating an unusual type of post-replicative DNA modification whose site-specificity would appear to be determined at the level of the DNA by much more extensive amounts of sequence. The modification system is specified by at least two members of the genus Streptomyces: Streptomyces lividans (2,3), which is commonly used for gene manipulation, and Streptomyces avermitilis (4), a commercial producer of antihelminthic avermectins. The hallmark of DNA isolated from these species is that it is susceptible to in vitro strand cleavage at the site of the modifications during gel electrophoresis. The cleavage reaction requires formation at the anode of an oxidative derivative, believed to contain a peracid moiety, of the common biological buffering compound Tris (5,6). This compound reacts with the modifications, which usually occur site-specifically at closely-opposed guanines on both strands, and consequently double-strand cleavage results. The reaction products are therefore discrete DNA fragments. The novelty of this reaction, and the fact that the same modifications do not react with piperidine, both indicate that the structure of the modified guanine is biologically unprecedented. Ultimately, this must await confirmation after purification and structural analaysis of the nucleotide, although as yet this has not proved possible. The modifying activity only partially modifies the DNA in vivo and to a variable extent at different sites, so that taking into account the site-specificity, the modification is estimated to represent [le]0.1% of total base composition. The low abundance of the species, possible co-elution with normal bases during chromatography and chemical instability are all factors which are believed to have obstructed its purification to date. Gaining more insight into the nature of the site-specificity of modification should enable its eventual enrichment.

Our previous studies investigating the site-specificity of modification concentrated on the preferred modification site of plasmid pIJ101, located in an intergenic region (7). Authentic modification at this site occurs within a 6 bp palindrome, itself part of a 13 bp repeat sequence, and requires extensive amounts of flanking sequence to either side. Both flanking sequences contain a copy of the 13 bp repeat in direct orientation, overlapping long inverted repeats, suggesting a complex interaction between the DNA substrate and the modifying activity. We have now investigated if the same pertains with other modification sites located in a high copy-number chromosomal DNA element: the 5.7 kb amplified DNA sequence (ADS_5.7) found in chloramphenicol-sensitive arginine auxotrophic mutants of S.lividans (8,9). We have mapped and extensively analysed the substrate requirements for modification at the preferred sites in this molecule.

Bacterial strains and plasmids

Escherichia coli strain F^-Z^-[Delta]M15 (10) was routinely employed as a host for plasmids used and constructed in this study. DNA modification was assayed after transformation and reisolation of plasmid DNA from S.lividans 66 (provided by D.A.Hopwood, John Innes centre, Norwich, UK). Native ADS_5.7 DNA was obtained from an S.lividans mutant strain E20 (9). The principal plasmids which were employed in the study are presented in Table 1.

Media, transformation and growth conditions

Escherichia coli strains bearing recombinant plasmids were cultured on L broth or plated on L agar (11), supplemented with appropriate antibiotics (25 µg/ml kanamycin, 50 µg/ml ampicillin, 10 µg/ml gentamicin). [alpha]-complementation by recombinant plasmids was tested by plating transformants on media containing 20 µg/ml Xgal. Plasmids were introduced by transformation using a standard calcium chloride-mediated procedure (11). Streptomyces strains were routinely grown on SFM agar, containing 20 g/l soya flour and 20 g/l mannitol, supplemented with appropriate antibiotics (concentrations as above, and thiostrepton 25 µg/ml). Plasmid or chromosomal DNA was isolated from cultures grown in tryptic soy medium (9). Transformation of Streptomyces involved polyethylene glycol mediated uptake of plasmid DNA by protoplasts (12), which were subsequently regenerated on RM14 medium (13) prepared as described previously (14).

DNA isolation and manipulation

Plasmid DNA was isolated from E.coli by the alkaline lysis technique (15), and typically from Streptomyces by a modified version of this technique (12). Prior to sequencing and primer extension assays, the DNA was further purified by precipitation. The DNA was then resuspended and further processed using Wizard plasmid purification columns according to the manufacturer's instructions (Promega). Streptomyces chromosomal DNA was purified as previously described (9). Plasmid DNA derived from E.coli was routinely visualised after electrophoresis in 0.8-1.2% agarose gels prepared in TBE buffer (11). For DNA isolated from Streptomyces, however, a HEPES-based buffer (5) was employed to avoid Tris-dependent DNA cleavage. DNA restriction and modifying enzymes were obtained from New England Biolabs or Life Sciences. To obtain ADS_5.7 restriction fragments in a sufficiently pure form for end-labelling prior to chemical sequencing, they were usually electrophoresed in two dimensions prior to gel elution. The latter was done using the Geneclean method as recommended by the manufacturer (BIO 101). Fragments were 3[prime]-end-labelled using Klenow fragment and the appropriate [[alpha]-³²P]dNTP, and 5[prime]-end-labelled, after dephosphorylation with alkaline phosphatase, using polynucleotide kinase and [[gamma]-³²P]ATP. For Southern hybridisation, DNA was transferred to Hybond N nylon membrane (Amersham) using a vacuum blotting system (Stratagene). An ADS_5.7 probe was prepared by labelling EcoRI-linearised pJD257 with digoxigenin-11-dUTP by random priming using a kit from Boehringer Mannheim. Hybridization conditions and subsequent detection by colour reaction with 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate were according to the manufacturer's instructions (Boehringer Mannheim).

Plasmid constructions

To clone the ADS_5.7, E20 chromosomal DNA was digested with BglII, which cuts once within each amplified unit, and separated by gel electrophoresis. After identifying the position of the 5.7 kb amplified sequence, a gel fragment was excised and the DNA subsequently eluted. The ADS_5.7-enriched fraction was then ligated to pUC8 (16) previously linearised with BamHI, and the ligation transformed into E.coli. Recombinants were identified after colony hybridisation with a probe consisting of a 1.1 kb SalI fragment obtained from pJD201 (17). pJD257, containing two copies of the 5.7 kb sequence ligated together in tandem repeat in the plasmid, was identified by its size after plasmid extraction from recombinants. Restriction fragments containing the four different modification sites were subcloned from pJD257 into the polylinker of pBGS19 (18), and thence as EcoRI-HindIII fragments into the corresponding polylinker of pUCS75 (19). In this manner a 685 bp SalI-BamHI fragment containing Site 1 was introduced into pUCS75 to give pUCS575, Site 2 was cloned as a 443 bp SalI-BamHI fragment contained in the pUCS75 derivative pTR15, Site 3 was cloned as a 702 bp SalI-PstI fragment and introduced into pUCS75 to give pUCS76, and Site 4 was cloned on a 755 bp BglII-SalI fragment into pUCS75, producing pAK2. Various deletion mutants of Sites 2, 3 and 4 were also constructed and introduced into pUCS75. Full details of these constructions are available on request.

Tris-dependent DNA cleavage, DNA sequencing and primer extension analysis

The nucleolytic activity was generated in standard Tris-acetate-EDTA (TAE) buffer containing 40 mM Tris, 20 mM sodium acetate, 0.8 mM EDTA, pH 7.5 adjusted with acetic acid. Conditions were employed which previously have been demonstrated to cause maximal DNA cleavage in the presence of saturating amounts of the nucleolytic activity (6). A 250 ml sample of buffer, maintained at 37°C, was electrophoretically activated by placing it in a horizontal submarine gel chamber (Bio-Rad) with platinum electrodes, and applying a constant voltage of 80 V. After 10 min activation, 500 µl of the buffer was sampled from ~5 mm adjacent to the anode and added to 0.5 µg DNA in 5 µl H₂O in a microfuge tube. The cleavage reaction was then allowed to proceed for 2 h at 37°C. The reaction was terminated by addition of 50 µl 3 M sodium acetate, pH 5 and 550 µl isopropanol. The DNA was precipitated at -20°C, prior to centrifugation, drying and resuspension of the pellet in 10 µl H₂O.

For chemical sequencing, four base-specific reactions on end-labelled DNA were employed (21) using dimethyl sulphate for base modification of guanine, formic acid for modification of both purines, and hydrazine with or without salt for modification of cytosines or both pyrimidines respectively, followed by piperidine-mediated strand scission of the modified bases. Primer extension and dideoxy-DNA sequencing reactions were performed with a CircumVent Thermal Cycle Dideoxy DNA Sequencing Kit largely according to the manufacturer's instructions (New England Biolabs). For reactions on templates cloned in pUCS75, standard 24mer forward and reverse sequencing primers were used. Primers were 5[prime]-end-labelled with [[gamma]-³³P]ATP and T4 polynucleotide kinase. In primer extension reactions, a deoxynucleotide mix consisting of 30 µM dATP, 100 µM dCTP, 100 µM dGTP and 100 µM dTTP substituted for the deoxy/dideoxy sequencing mixes provided in the kit. For thermal cycling, a `hot-top' Crocodile III thermal cycler (Appligene) was employed, using 20 cycles of 30 s at 95°C denaturing, 30 s at 55°C annealing, and 30 s at 72°C extension. Extension and sequencing products were separated using 8 and 6% (w/v) polyacrylamide denaturing sequencing gels prepared by appropriate combination of concentrate and diluent (Scotlab, UK). Autoradiographs were made by exposing X-ray film (Hyperfilm [beta]Max, Amersham) against the gel either together with an intensifying screen at -70°C to detect ³²P-labelled DNA, or at room temperature for ³³P-labelled extension products. They were then scanned with an HP ScanJet IICX and the signal intensity of specific bands quantified using Phoretix Life Software (Phoretix Ltd, Newcastle, UK). Loading variations between lanes were corrected according to the signal intensities of reference bands not arising due to Tris-mediated cleavage of the template. As an over-saturated exposure of the film could be formed as a result of high-frequency Tris-mediated cleavage, a series of shorter exposures was made to obtain a reliable estimate of the amounts of these products. The results presented represent mean values obtained from three independent DNA cleavage reactions on modified DNA (standard deviations were [le]11% of these values).

DNA sequences were analysed using the GCG package (22) provided by SEQNET at the Daresbury Laboratory, UK. The COMPARE program, employing a Word Comparison Algorithm, was used to search for regions of 4, 6, 8 or 10 bp perfect identity shared between the four ADS_5.7 sequences and the preferred modification site of pIJ101. The REPEAT program was used to find directly repeated sequences in each site examined, using window sizes of 6, 7, 8 or 10 bp. The STEMLOOP program was used to search for inverted repeats in each sequence, typically using initial parameters of a 12 bp minimum stem with 24 bonds, and loop sizes set at between 3 and 20 nt.

RESULTS

Mapping and chemical sequence analysis of modification sites in ADS_5.7

The locations of the four principal double-strand modification sites present in ADS_5.7 were established by isolating overlapping SalI, PvuII and SacII restriction fragments from total chromosomal DNA digests separated by electrophoresis, inducing Tris-dependent double-strand cleavage at the individual modification sites and visualising the fragmentation products after electrophoresis and Southern blotting, hybridising with an ADS_5.7-specific probe. As a negative control, similar fragments derived from the cloned ADS_5.7 in plasmid pJD257, passaged through E.coli, were treated under identical conditions. The positions of the modification sites were deduced by estimating the sizes of the cleavage products identified this way compared to molecular weight standards (results not shown). These results were subsequently confirmed and the locations more precisely determined by isolating smaller single-end-labelled restriction fragments, encompassing the individual modification sites (Fig. 1), and comparing the Tris-mediated cleavage products derived from these fragments with Maxam-Gilbert chemical sequence ladders obtained from similar fragments isolated from the E.coli plasmid pJD257. The success of this analysis depended upon the modification sites being in the vicinity of appropriate restriction sites both for end-labelling and, in some cases, for subsequent removal of one of two labelled ends. For a given modification site, this limited the scope for examination of both strands as a desired fragment would often comigrate with another amplified restriction fragment of similar molecular weight. It also placed a constraint on whether we could examine cleavage products with 5[prime] or 3[prime] termini resulting from Tris-mediated strand scission at the modified bases of each site. For at least one strand of each of the four sites, the locations of the modifications were determined by comparing with a sequence ladder 3[prime]-labelled strands with 5[prime] ends generated by Tris-mediated strand scission. In all four cases, however, the precise position of the modification could not be assigned unambiguously as there were evidently two cleavage products with different 5[prime] termini. This is exemplified in Figure 2A, which illustrates the 5[prime] termini produced by strand scission at the modification on the bottom strand of Site 4. For each of the four sites examined, there was a 2-4-fold greater abundance of the larger product, which differed from the smaller in terms of its electrophoretic mobility by ~1 nt. The 3[prime] termini resulting from Tris-mediated cleavage of at least one 5[prime]-labelled strand of Sites 1, 2 and 3 were also examined. Again, two products were observed after autoradiography, with the smaller being typically 3-fold more abundant than the larger, as illustrated for the bottom strand of Site 2 (Fig. 2B).

Figure 1. Map of the preferred modification sites of ADS_5.7. (A) The exact locations (in parentheses) of the modification and restriction sites refer to the GenBank deposited sequence, accession number U22894. The two ORFs are denoted by shading with arrows indicating their orientation: the lighter shaded ORF represents the putative chitinase gene, the darker shaded ORF encodes a DNA binding protein. (B) The regions of both strands analysed by chemical sequencing are indicated, with asterisks denoting the radiolabelled end of each strand. (C) The origins and sizes of the cloned restriction fragments containing the individual modification sites, together with the plasmid number of each respective pUCS75 derivative are indicated.

Figure 2. Analysis of the positions of Tris-dependent cleavage within native amplified restriction fragments. (A) Analysis of a 3[prime] labelled HinfI-SalI fragment containing Site 4. (B) Analysis of a 5[prime] labelled SalI-BamHI fragment containing Site 2. Lanes labelled + and - contain DNA derived from S.lividans strain E20, treated with or without activated Tris respectively. Lanes C, Y (pyrimidines), R (purines) and G are the products of chemical sequence reactions on a similar fragment isolated from pJD257 passaged through E.coli. Solid arrows indicate the position of the major product, and open arrows the minor product, resulting from Tris-dependent cleavage at the modified base of the bottom strand of the two sites.

Figure 3. Positions of DNA modification in deletion mutants of Sites 2, 3 and 4. The extent and coordinates, in square brackets, of remaining sequences in deletion mutants of each of the modification sites are indicated, together with the corresponding plasmid number of the respective pUCS75 derivative. The smallest extent of sequence required for authentic modification at each site is represented for comparison. Solid and open triangles represent, respectively, the positions of frequent and infrequent modification detected using the primer extension assay. Figures in parentheses are quantitative estimates of the frequencies of modification. These figures are ratios for the amount of extension product obtained with Tris-reacted and untreated templates after correction for gel loading differences; a ratio of 1 indicates no detectable cleavage and hence little or no base modification.

To aid further analysis of the different modification sites, each one was cloned on a discrete restriction fragment (Fig. 1) into the polylinker of plasmid pUCS75. The resulting recombinant plasmids were passaged through S.lividans, reisolated and restricted with EcoRI and HindIII to cut out the cloned region. These fragments were isolated in parallel with the original fragments derived directly from the ADS_5.7, treated with activated Tris, and the fragmentation products visualized after gel electrophoresis and Southern hybridisation. This confirmed the occurrence of modification within each cloned fragment in its new context (results not shown). In each case the expected products were observed, although the restriction fragment representing the cloned copy of Site 2 gave two additional fragments, consistent with cleavage at a secondary adjacent modification site (see below). While these extra products were discernible in the case of the corresponding ADS_5.7 derived fragment, they were clearly more abundant in the cloned copy. Further analysis of the locations of each site and the extent of sequences determining site-specifity in vivo could now be addressed with the cloned copies. This analysis was performed for Sites 2, 3 and 4, and involved use of a primer extension assay to locate modified bases and construction of deletion mutants with varying extents of sequences flanking modification sites.

Modification site 2

Site 2 was initially cloned on a 443 bp BamHI-SalI fragment. The resulting recombinant plasmid pTR15 was passaged through S.lividans, plasmid DNA isolated and an aliquot treated with activated Tris. Both Tris-treated and untreated DNA were employed in primer extension reactions using pUC/M13 forward and reverse primers. The results of the primer extension analysis on full-length and deletion mutants of Site 2 are summarised in Figure 3; the profile for pTR15 was similar to that indicated for pUCS99. Reverse primer (RP) extension with the Tris-treated template revealed specific termination opposite the third guanine in a GpGpGp sequence (Fig. 4A). This is consistent with modification of the second guanine [nucleotide 6826 of the top strand in the GenBank deposited sequence of the Amplifiable Unit of DNA, AUD1, accession number U22894 (23)] in vivo, and subsequent strand scission releasing the template strand with the third guanine as the 5[prime] nucleotide. This was one of the reaction products observed with chemical sequencing, the other containing an additional 5[prime] group. Another termination product of approximate equal intensity was observed for the same template opposite the third guanine of another GpGpGp sequence, corresponding to modification of the guanine residue on the top strand at position 6665 (Fig. 4A). Only limited Tris-mediated cleavage at this position was observed on the same strand of the chromosomal-derived fragment (Fig. 4B). Two products with different 3[prime] termini were observed, the smaller being consistent with strand scission and [beta]-elimination at the guanine residue at coordinate 6665. Moreover, primer extension with the forward primer (FP) with the pTR15 template resulted in an abundant specific termination product consistent with modification on the bottom strand at position 6668; again chemical sequencing of the corresponding chromosomal-derived strand revealed only limited strand scission at this position. Densitometric analysis of both the Southern blot (see above) and autoradiographs comparing Tris-dependent cleavage products indicated that the cloned copy of this region appears to undergo a 4-5-fold increase in modification frequency at positions 6665 (top strand) and 6668 (bottom strand) in relation to the sequence in its normal context. FP extension with the pTR15 template also revealed an abundant termination product consistent with modification at a guanine residue at position 6828 of the bottom strand. The major product after Tris-mediated cleavage of the same 5[prime]-end-labelled strand and analysed in comparison with a chemical sequence ladder was also consistent with modification and subsequent [beta]-elimination of this residue, in that it had the same mobility as the product of piperidine cleavage at the base in the Maxam-Gilbert sequencing reaction (Fig. 2B). To discriminate between the two sites, the modifications on opposite strands at positions 6665 and 6668 are subsequently referred to as Site 2B, whereas those at positions 6826 and 6828 constitute Site 2A.

Figure 4. Comparison of DNA modification within the native and cloned Site 2 region. (A) RP primer extension and cycle sequencing products obtained with the pTR15 template, containing the cloned 443 bp BamHI-SalI fragment encompassing Site 2. Lane + contains extension products obtained with template DNA purified from S.lividans and subsequently treated with activated Tris. Arrows indicate the specific termination products resulting from Tris-dependent cleavage of the template (top) strand at Sites 2A and 2B. Dideoxy sequencing products (T, G, C and A) were obtained with the template purified from E.coli. (B) Analysis of the same region of the same strand of the DNA from its native context, after 5[prime]-end-labelling the BamHI site. Lanes + and - contain DNA treated and untreated with activated Tris, respectively. The positions of Tris-dependent cleavage at Sites 2A and 2B, in opposite orientation with respect to (A), are indicated by arrows.

To define further the extent of sequences required for modification at Site 2A, a 28 bp linker containing Site 2A sequences extending from coordinate 6816 to 6843 was synthesised and cloned into plasmid pUCS75. Tris-treated DNA, isolated after passaging the recombinant plasmid pTR30 through S.lividans was subjected to primer extension analysis using both forward and reverse primers. However, no termination products could be detected within this cloned region, suggesting that it was not recognised in vivo by the modifying activity. Consequently, deletions of both left (LH) and right hand (RH) sequences flanking Site 2A present in plasmid pTR15 were constructed and analysed by primer extension. Plasmid pUCS99, containing a deletion of 32 bp of distal RH flanking sequence, was found to undergo a similar extent and pattern of modification to that of pTR15. Subsequently, Site 2A was cloned as a 121 bp AvaI-SmaI fragment, containing 88 bp of LH flanking sequence, excluding Site 2B, and 31 bp of RH flanking sequence. FP extension on the resulting plasmid pUCS983 revealed abundant termination consistent with modification at position 6828 on the bottom strand, whereas for RP extension termination was barely detectable at the expected position. This suggested that as a result of the deletion of flanking sequences, only one strand of Site 2A underwent efficient modification in vivo.

A truncated version of Site 2B, divorced from Site 2A, was also tested for modification. This sequence contained 111 bp of LH and 65 bp of RH flanking sequence. Primer extension analysis on this template plasmid pUCS993 revealed termination products consistent with modification on opposite strands at positions 6665 and 6668, as had been observed for both pTR15 and pUCS99.

Modification site 3

Site 3 was initially cloned as a 702 bp PstI-SalI fragment in plasmid pUCS76. Several deletion mutants containing varying extents of the LH and RH flanking sequences were constructed and assayed for modification, as was a synthesised cloned linker containing just 24 bp of the region. The results are summarised in Figure 3. The smallest deletion mutant in which authentic modification was observed, and represented by plasmid pUCS92, contained 58 bp of LH and 112 bp of RH flanking sequence. For this plasmid, and for all the other plasmids tested containing more extensive sequences, the observed termination products for FP (Fig. 5A) and RP extension were consistent with modification at guanine residues located on opposite strands at positions 7991 and 7993, respectively. Modification at these positions, followed by Tris-mediated strand-scission involving [beta]-elimination of both phosphates, would give the smaller products observed in chemical sequence analysis of both strands of Site 3. In contrast, no termination products were detected within the 24 bp synthetic sequence cloned in plasmid pUCS96, indicating that this sequence is not modified in vivo. Suprisingly, quite different patterns of modification were detected in mutants containing intermediary extents of Site 3 compared to either pUCS92 or pUCS96. The largest of these cloned regions, represented by plasmid pUCS106, contained in comparison with pUCS92 just 28 bp, as opposed to 58 bp, of LH flanking region, but the same extent of RH flanking sequence. Only a minor termination product was detected by FP extension (Fig. 5C), consistent with limited modification of the guanine residue at 7991, and no modification of residue 7993 on the opposite strand was observed when RP extension was performed. A minor termination product was detected, however, with RP extension, consistent with single-strand specific modification at a guanine residue at position 8035.

Figure 5. Deletions of flanking sequences cause changes in the positions and frequency of modification at Site 3. FP extension and dideoxy sequencing reactions were performed on plasmids pUCS92, containing the minimum sequence to direct authentic modification at Site 3 (A), pUCS107, containing a 45 bp deletion of RH flanking sequences (B), and pUCS106, containing a 30 bp deletion of LH flanking sequence. Lanes + and - represent extension on plasmid obtained from S.lividans and incubated with or without activated Tris respectively. The solid arrow indicates the position of the extension product resulting from Tris-dependent cleavage at the normal position (7991) for modification on the top strand. Very little termination was detected at this position for plasmids pUCS107 and pUCS106. The open arrow indicates the position of the extension product resulting from Tris-dependent cleavage at the displaced single-stranded modification (7969) in plasmid pUCS107.

Another deletion mutant, represented by plasmid pUCS107 and containing a `complete' LH flanking sequence of 58 bp, but only 67 bp as opposed to 112 bp of RH sequence, underwent efficient modification at residue 7993 on the bottom strand, as well as a limited modification at residue 8035 of the same strand, as had been observed in pUCS106. FP extension revealed only limited modification at position 7991 on the top strand, but, remarkably, a strong termination product was observed consistent with efficient single-strand-specific modification of the guanine at position 7969 (Fig. 5B). For plasmid pUCS93, containing only 21 bp of RH sequence but a full 58 bp LH sequence, no modifications were detected on the bottom strand using RP extension. With FP extension, two approximately equimolar termination products were obtained, consistent with modification occuring at adjacent guanine residues on the top strand at positions 7990 and 7991.

Modification site 4

The results from primer extension analysis on plasmids containing varying extents of Site 4 are presented in Figure 3. Chemical sequence analysis had provided information on the location of the modification on only the bottom strand of Site 4. For similar analysis of Sites 2 and 3, the smaller of the two products with 5[prime] termini at the position of Tris-mediated cleavage could be attributed to [beta]-elimination at the site of modification. Following from this, the principal DNA cleavage site of the bottom strand was deduced to be the guanine at position 9700. This was confirmed by FP extension on Tris-treated plasmid pAK2, containing the cloned copy of site 4 on a 755 bp BglII-SacI fragment. However, another termination product was observed consistent with less efficient modification of another guanine residue on the bottom strand at position 9696. RP extension on the Tris-treated template resulted in two approximately equimolar termination products consistent with modification at positions 9697 and 9698 on the top strand. A similar modification pattern was observed for plasmid pNH2, in which 167 bp of RH flanking sequence had been deleted. Deletion of a further 227 bp of the RH flanking sequence as in plasmid pAH2, however, had a dramatic affect on the pattern of modification. On the top strand only limited modification of guanine 9698 was detected, and no modification of guanine 9697 was observed. A more pronounced modification was instead detected at a guanine residue at position 9744. FP extension revealed modification at several positions on the bottom strand: single-strand-specific modification of guanine 9600, modifications at positions 9696 and 9700 as observed for the undeleted cloned region, and modification of a guanine at 9742 closely opposed to the displaced modification observed on the top strand of this mutant.

The pattern of modification on the remaining sequences of a further deletion mutant, plasmid pAK4 containing only 19 bp of RH flanking sequence, resembled that of pAH2. FP termination products were consistent with single-strand-specific modification at position 9600, and also modification of guanines 9696 and 9700. RP extension resulted in limited termination consistent with some modification of residue 9698 on the top strand. The two smallest Site 4 mutants analysed, represented by plasmids pAB2 and pADS2 and containing 25 or 7 bp of LH flanking sequence, and 19 or 9 bp of RH flanking sequence, respectively, underwent similar modification in vivo. No modification was detected in the top strand, whereas the guanines at position 9700, and to a limited extent 9696, on the bottom strand were modified.

DISCUSSION

The principal modification sites located within the ADS_5.7 occur in either of the two open reading frames: Site 3 in a gene encoding a DNA binding protein known to be necessary for amplification to occur, and the other sites located in a putative chitinase gene (23). In this respect, the sites differ to the preferred site of pIJ101 which is located within an intergenic region. Significantly, there is also a difference with regard to the extent of modification that the intragenic and intergenic sites undergo in vivo. Although the primer extension assay we employed provided only indirect quantification of the degree of modification, comparison between sites revealed that the pIJ101 sequence is modified approximately 10 times more efficiently than the principal sites within the ADS_5.7 (7). It was therefore informative to compare features which the various sites share in common and note the distinctive aspects of the pIJ101 site which could render it more sensitive to in vivo modification. To carry out this comparative analysis, we considered firstly the nature of the sequences immediately surrounding the closely opposed modifications, subsequently referred to as `core' sequences, and secondly the characteristics of the flanking sequences defined by deletion analysis as being crucial for authentic modification within the core sequences.

Figure 6 represents an alignment of the core sequences of the various sites. As we present evidence that each strand may be modified independently (see below), and because of the asymmetry of the sequences, both strands of the respective sites are compiled. A core sequence of 8 nt is well conserved between sites; the significance of other conserved bases to either side is unclear, given the overall 75% G+C content of Streptomyces DNA. The consensus 8 nt sequence is in fact palindromic. The modifying activity was observed to act on either, and occasionally both, of the two central guanine residues in different sites and to modify a guanine on the opposite strand separated by a distance of 2 or 3 bp. Similar variance in the stagger between modified bases on opposite strands was observed in the pIJ101 site (7). Moreover, the central 6 bp palindrome found in the pIJ101 site is also a feature of Sites 1 and 4. With 10 out of 13 bp homology shared between the bottom strand and the central 13 bp repeat of the pIJ101 site, Site 1 appears to be the most closely related overall. However, for all the core regions of the ADS_5.7 sites, including Site 1, the greatest degree of shared homology is within the 8 bp sequence.

Figure 6. Comparison of the core sequences of different modification sites. (A) Both top and bottom strands of the core sequences of different modification sites are aligned around a highly conserved central 5[prime] GGCC sequence. The modified guanines for each sequence are indicated in italicised bold face. The extent of the central 13 nt direct repeat (top strand) of the preferred pIJ101 site is indicated by the arrow. A consensus sequence (B) derived from this comparison is presented; the positions for potential modification at the two central guanine residues are indicated by shading, and the 8 nt palindrome is underlined. (C) The sequences of displaced double-strand modification, Site 4* (9744/9742), observed in the Site 4 deletion mutant pAH2, and displaced single-strand modification in deletion mutants of Site 3 (pUCS107) and Site 4 (pAH2 and pAK4), are aligned with the consensus.

Efficient and authentic modification within the core sequence of the pIJ101 site requires 70-80 bp of LH and RH flanking sequence which include a symmetrical arrangement of repeat sequences. The site contains the three 13 bp direct repeats with two flanking inverted repeats overlapping the left and right direct repeats. While deletion analysis of the ADS_5.7 sites was not exhaustive, it demonstrated a similar requirement for extensive sequences surrounding the core sequence. For example, the smallest cloned region directing authentic modification of Site 3 was 172 bp long. The flanking sequences of these sites were scrutinised using GCG package programmes (22) which revealed: (i) no significant DNA homology shared between the flanking sequences of different sites; (ii) the absence of significant inverted repeats in any of the sequences; and (iii) no direct repeats >6 bp in length shared between all sites and, in particular, no good matches to the 13 bp repeats of the pIJ101 site. There was no common pattern in the way in which short direct repeats [le]6 bp were distributed when comparing the overall architecture of the different sites. The only significant homology shared between all sites was confined to the core sequences. These analyses support a model in which the repeat structures present in the pIJ101 site could contribute to the greater susceptibility to in vivo modification this site exhibits compared to the ADS_5.7 sites. As we have previously suggested, DNA topology could influence the ordering of these repeats and hence modification. Likewise, the flanking sequences of the ADS_5.7 sites could influence local DNA topology and affect modification within the respective core sequences. Differences in superhelical density could also explain the observed increase in the degree of modification at Site 2B as a result of moving it from its normal context within the ADS_5.7, which is chromosomal, into plasmid pUCS75.

Another feature revealed by analysis of the pIJ101 site was displaced double-strand modification, albeit comparatively inefficient, within the RH direct repeat as a consequence of deletion of the LH repeats (7). Deletion of flanking sequences in the ADS_5.7 sites also resulted in displaced modification, frequently on only one strand, but for Site 4 also closely-opposed on both strands. Alignment of the sequences where these displaced modifications occur revealed that whereas the displaced double-strand modification sequence resembled the consensus core sequence, the sequences where displaced single-strand-specific modification occurred were quite divergent (Fig. 6). Predominant modification of just one strand within the core sequence was also a frequent feature of the deletion mutants. These results suggest that the modifying activity can act on one strand alone; infrequent single-strand-specific modification had not previously been detected using a less sensitive assay (3).

Whereas the locations of the DNA cleavage sites could be determined unambiguously by primer extension, analysis of native Tris-cleaved amplified DNA fragments alongside chemical sequencing reactions revealed in each case two different 5[prime] and 3[prime] termini. One terminus was consistent with strand scission involving [beta]-elimination of both phosphates from the sugar of the same modified base identified by primer extension, releasing fragments with 3[prime] and 5[prime] phosphate groups (similar to the outcome of chemical sequencing reactions). The electrophoretic mobility of fragments with alternative termini was consistent with either reactivity of the same modified guanine and formation of fragments terminating with bulkier 3[prime] and 5[prime] functionalities, or modification and subsequent strand scission at the next guanine residue. For the undeleted sites analysed by primer extension, with the exception of the top strand of Site 4, there was no evidence for modification at adjacent guanines. This might have been obscured if each DNA molecule was coincidentally modified and subsequently cleaved at both guanines. However, the relative abundance of molecules with alternate 3[prime] and 5[prime] termini clearly showed this not to be the case. This suggests that the chemistry of Tris-dependent cleavage at the modified bases is more complex than originally assumed, with the release of fragments with terminal phosphate groups being only one outcome. This is reminiscent of the mixture of products generated due to chemical instability of bleomycin-induced abasic sites (24). Two different 3[prime] termini produced as a result of strand cleavage induced by incubation with alkali or aqueous alkylamines have been characterised as consisting of either a phosphate group or a 2,4-dihydrocyclopentenone moiety. Interestingly, these abasic sites are also known to be unstable during electrophoresis in Tris-buffers (25), although, to our knowledge, the terminal functionalities present in the resulting cleaved products have not been examined. We have previously proposed that abasic sites may be intermediates generated by the reaction of activated Tris with the modified guanines in Streptomyces DNA (6). Further evidence for this must await chemical characterisation of the modified base, but detailed analysis of the terminal functionalities resulting from Tris-dependent cleavage may also help to define the nature of the reaction.

Analysis of the extent of the ADS_5.7 sites underlines the unusual complexity defining the site-specificity of the modifying activity first revealed by characterisation of the preferred pIJ101 site. We are continuing to elucidate the role of the repeat structures and DNA topology which probably contribute to enhanced modification at the latter site. These structures may be characteristic to intergenic sites and consequently play a role in the observed affect of this DNA modification on gene expression (X.Zhou and T.Kieser, personal communication).

ACKNOWLEDGEMENTS

T.R. was supported by a studentship from SERC. A.B. was supported by the University of Nigde, Turkey. The work was initiated by P.D. in the laboratories of Professor Hildgund Schrempf, Munich, Germany, and Professors Julian Davies and Charles Thompson, Pasteur Institute, Paris.

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*To whom correspondence should be addressed. Tel: +44 1792 295667; Fax: +44 1792 295447; Email: p.j.dyson@swansea.ac.uk
Present addresses: ⁺University of Nigde, Nigde, Turkey and ^§Cytocell Ltd, Banbury, Oxfordshire, UK

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