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• W1979348985 abstract "Escherichia coli plasmid DNA activated for initiation of duplication is in a stable low linking number supercoiled conformation. Low linking number DNA is methylated at the internal purines of a frequent 5′-Pyr-Pyr-Pur-Pur tetramer with a 5′-Pyr-Pur-3′ axis of symmetry and is cut at the axis of symmetry by pneumococcal restriction enzyme DpnI when methylated in both strands. Purine methylation is of adenine in one strand and guanine in the other. Methylation of one of the two purines is removed during the cell cycle, presumably before the reverse shift to the B-supercoiled conformation. The topological transition was reconstituted in vitro only with DNA unmethylated at purines. Methylation-restriction analyses coupled with the chemical properties of low-linking number DNA and B-DNA respectively, suggest that removal of guanine methylation is essential for the low-linking number to B-DNA transition and hence for the deactivation of replication. Demethylation of methylguanine could explain the presence in E. coli of the two-member inducible operon known as ada. Characteristics of ada suggest a cascade of chemical DNA modifications that reverse prereplicative guanine methylation. Guanine demethylation could provide a model for the pivotal role played by de novo methylation in replication and for the essential role of “repair” enzyme ExoIII in demethylation leading to the reversal of replicative DNA activation and other processes that affect DNA function. Escherichia coli plasmid DNA activated for initiation of duplication is in a stable low linking number supercoiled conformation. Low linking number DNA is methylated at the internal purines of a frequent 5′-Pyr-Pyr-Pur-Pur tetramer with a 5′-Pyr-Pur-3′ axis of symmetry and is cut at the axis of symmetry by pneumococcal restriction enzyme DpnI when methylated in both strands. Purine methylation is of adenine in one strand and guanine in the other. Methylation of one of the two purines is removed during the cell cycle, presumably before the reverse shift to the B-supercoiled conformation. The topological transition was reconstituted in vitro only with DNA unmethylated at purines. Methylation-restriction analyses coupled with the chemical properties of low-linking number DNA and B-DNA respectively, suggest that removal of guanine methylation is essential for the low-linking number to B-DNA transition and hence for the deactivation of replication. Demethylation of methylguanine could explain the presence in E. coli of the two-member inducible operon known as ada. Characteristics of ada suggest a cascade of chemical DNA modifications that reverse prereplicative guanine methylation. Guanine demethylation could provide a model for the pivotal role played by de novo methylation in replication and for the essential role of “repair” enzyme ExoIII in demethylation leading to the reversal of replicative DNA activation and other processes that affect DNA function. The replication studies reported here and in Ref. 1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar deal with the miniplasmid RepFIC, isolated from Escherichia coli pathogen 307 (2Saadi S. Maas W.K. Hill D.F. Bergquist P.L. J. Bacteriol. 1987; 169: 1836-1846Crossref PubMed Google Scholar). The RepFIC origin is prototypic of pathogenic plasmids of E. coli, Salmonella, and Shigella. The four peptides expressed by mini-RepFIC are essential and sufficient for the in vivo once/cell cycle activation associated with replication (3Maas R. Wang C. J. Bacteriol. 1997; 179: 2163-2168Crossref PubMed Google Scholar, 4Maas R. Wang C. Maas W.K. J. Bacteriol. 1997; 179: 3823-3827Crossref PubMed Google Scholar). The minimal replicon used in these studies is 3000 base pairs in size and fully regulated, with each plasmid unit essentially replicating once during the cell cycle. In a previously reported optimal recovery of transformants approximately every plasmid molecule transferred by electroporation initiates replication (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Such conditions “imprint” the altered L-DNA conformation of plasmid maintenance, characterized by lowered linking number or helical density (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The imprinting of L-DNA maintenance led to its isolation in pure form and facilitated its characterization. Thus, it could be established by electron microscopy that L-DNA is intact and supercoiled and has significantly lowered solubility in aqueous solutions as well as altered electrophoretic mobility (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). L-DNA has sometimes been overlooked because it partitions into phenol chloroform. The altered conformation is imprinted under specific circumstances at the time of transformation for other E. coli plasmids such as pBR, mini-F, and cloned OriC. Exclusive maintenance of DNA in the L-conformation results in filamentous growth, suggesting inhibition of cell division when L-DNA does not revert to B. Thus, the topological transition of L-DNA to B-DNA is of importance for optimal growth and suggests that the transition was selected in the course of evolution. As expected of a plasmid activated for replication, RepFIC plasmid in the L-conformation efficiently transforms E. coli hosts. When associated proteins including the DNA-methyltransferases have been stringently removed from transferred L-DNA, transformation efficiency decreases by 5 orders of magnitude (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Efficiency is restored by activation of the methyltransfer metR regulon after DNA entry, emphasizing the importance of de novo methylation to replication (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The interpretation derived from the previous work is that initiation of replication is associated with a significant increase in the level of methylation. Whether de novo methylation is retained or not in the parental strands after DNA duplication was not addressed in Ref. 1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar and has been partially analyzed in this work. Prevention of rapid remethylation (5Boye E. Lobner-Olesen A. Cell. 1990; 62: 981-989Abstract Full Text PDF PubMed Scopus (175) Google Scholar, 6Russell D.W. Zinder N.D. Cell. 1987; 50: 1071-1079Abstract Full Text PDF PubMed Scopus (157) Google Scholar), related to the removal of protein initiator and methyltransferases from the DNA by Lon protease, contributes to the prevention of a second round of replication (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). L-DNA is methylated in both strands by dam-methyltransferase at frequent intervals both in vivo and in vitro at sequences different from GATC (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The methylated tetramers are here identified and shown to be methylated at adenine in one strand and guanine in the other. The obligatory assortment of parental strands then results in tetramers that are alternatively methylated at adenine or guanine in each transmitted parental strand (7Meselson M. Stahl F.W. Proc. Natl. Acad. Sci. U. S. A. 1958; 44: 671-682Crossref PubMed Google Scholar). Analysis of purine methylation is consistent with one of the two bases being demethylated, in all probability methylguanine. The phenotype of conditional ExoIII mutants of E. coli led to the educated guess that ExoIII conforms L-DNA to B in the cell. ExoIII in fact catalyzed in vitro the L to B transition in the absence but not in the presence of purine methylation. Bacterial Strains and Plasmids—The following E. coli strains were used as hosts: C600 (thi-1 thr-1 leuB6 lacY1 tonA21 supE44 mcrA), JT4000 (Δlon-510), GM3819 (dam-16::KanR), GM31 F- (dcm-6), and GM2198 (dam-13::Tn9 dcm-6). The miniplasmid used, pRM3930, consists of the RepFIC basic replicon and the streptomycin-spectinomycin resistance cassette Ω. Growth of Bacteria Cultures—Cultures bacteria were grown in tryptone-yeast extract medium (Difco), with added 0.1% glucose, and 50 μg of spectinomycin/ml. Other supplements are described in the figure legends. Preparation of Plasmid DNA and Restriction Analysis—DNA was prepared with Qiagen plasmid mini Kits as recommended by the manufacturer. Associated proteins were then removed by “salt stripping” as previously described (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The enzymes were purchased from Roche Applied Science (EcoRII) and New England Biolabs. The reaction volumes were 10 μl/lane, containing four to five units of enzyme. DNA analysis was by 0.8% agarose gel electrophoresis. ExoIII Reactions—ExoIII was purchased from New England Biolabs. ExoIII does not degrade circular intact DNA (8Kornberg A. Baker T. DNA Replication. 2nd Ed. W. H. Freeman and Company, New York, NY1992Google Scholar), making the basis for enzyme units described in the New England Biolabs catalog irrelevant to this work. ExoIII exhibited topoisomerase activity toward L-DNA and presumably bound to cognate loci, calculated to be at least 39 in number/replicon sequence. Fifty New England Biolabs units/lane were used and found to be sufficient. The enzyme was “inactivated” as recommended by the manufacturer at 70 °C for 20 min. The reaction volumes were 10 μl/lane. Newly Characterized Methylation of A, G, and C in Replicon DNA—The replication switch-off associated with methylation of one strand and seen in the transformation of dam- hosts with methylated plasmids (6Russell D.W. Zinder N.D. Cell. 1987; 50: 1071-1079Abstract Full Text PDF PubMed Scopus (157) Google Scholar) is observed with RepFIC only when dam-dcm- hosts are transformed. This suggests that dam-methyltransferase (dam-Mtase) 1The abbreviations used are: Mtase, methyltransferase; ds, double-stranded; MMS, methyl methanesulfonate. and dcm-methyltransferase (dcm-Mtase) of E. coli, contrary to current dogma, methylate a common sequence. The cutting activity of restriction enzymes is recognized in vitro by the electrophoretic separation of resulting double-stranded (ds) fragments. ds fragments are obtained when the cognate restriction sequence has an axis of symmetry (8Kornberg A. Baker T. DNA Replication. 2nd Ed. W. H. Freeman and Company, New York, NY1992Google Scholar). The pneumococcal enzyme DpnI makes blunt cuts at 5′-meA↓T in GATC. dam-Mtase methylates the As in both strands. When DpnI was used to restrict RepFIC L-DNA, a 2.5-kb fragment with no GATC sequences was shredded to fragments <100 base pairs in length (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), suggesting that DpnI cuts DNA at additional loci different from 5′-meA↓T. The most numerous alternating Pyr-Pyr-Pur-Pur loci by a factor of ∼10 in RepFIC are 5′-CCAG and its complementary sequence 5′-CTGG. Some of these loci were first identified serendipitously by piperidine digestion (9Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 499-560Crossref PubMed Scopus (9015) Google Scholar) in a control experiment that will be described briefly below. Methylation is at A in 5′-CCAG and the opposite internal G, respectively (see Fig. 2B). The DpnI cuts are inferred to be at C↓meA and T↓meG (Table I) with an axis of symmetry at the blunt cut.Table IThe dam- and dcm-methyltransferases of E. coliSequence methylatedDNA conformationMethylation typeRestriction enzymeMethylationRestriction cutsEssentialBlockedDamGATCCanonicalDpnIYesGA ↓ TCCT ↑ AGDamCCAG-3′LDe novoDpnIYesCC ↓ AG-3′GGTC-5′GG ↓ TC-5′EcoRIIYesC ↓ CAG-3′DcmCCAG-3′CanonicalEcoRIIYesC ↓ CAG-3′GGTC-5′DamCCAGG-3′LDe novoEcoRIIYesC ↓ CAGGCGTCC-5′GGTC ↑ CDcmCCAGG-3′CanonicalEcoRIIYesC ↓ CAGGGGTCC-5′GGTC ↑ C Open table in a new tab 5′-CCAG and its complement is part of the 5-mer sequence 5′-CCWGG (where W = A or T), restricted at CC of each strand by EcoRII in the absence of cytosine methylation (10Palmer B.R. Marinus M.G. Gene (Amst.). 1994; 143: 1-12Crossref PubMed Scopus (222) Google Scholar). Because the pentamer sequence is methylated by dcm-Mtase (10Palmer B.R. Marinus M.G. Gene (Amst.). 1994; 143: 1-12Crossref PubMed Scopus (222) Google Scholar), investigation of purine methylation of the sequence demands the absence of cytosine methylation. 5′-CCWGG occurs eight times in RepFIC, four times in one orientation and four times in the other (see Fig. 2). MvaI cuts 5′-CCWGG regardless of methylation. In the gel of Fig. 1, plasmid isolated from a Dam+Dcm+ host was restricted by MvaI (lane 2) and not by EcoRII (lane 3). Thus, in the presence of both methyltransferases, methylation prevents EcoRII restriction of all loci. The calculated length between loci 6 and 7 (Figs. 1 and 2) is 1258 base pairs, providing a convenient diagnostic fragment for restriction at these two loci. The diagnostic fragment was indeed obtained by digestion with MvaI and is indicated in lane 2 of Fig. 1. Plasmid isolated from a Dcm+dam- strain is equally resistant to digestion by EcoRII (lane 7). Thus, cytosine methylation is sufficient for blocking EcoRII restriction. EcoRII treatment of completely unmethylated DNA produced an extensive smear, suggesting numerous previously unknown methylation loci as already proposed (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) and an extended specificity for EcoRII restriction outside the 5′-CCWGG pentamer (lane 5). Similar smears are seen when plasmid DNA is treated with pancreatic DNase, an enzyme that makes single-stranded cuts in ds DNA. They are also seen when DNA methylated at purines is treated with piperidine. In this second case single-stranded cuts form the basis for Maxam-Gilbert sequencing (9Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 499-560Crossref PubMed Scopus (9015) Google Scholar). The sequence 5′-CCWGG contains a 5′-CC doublet in each strand. The simplest explanation for the single-stranded cuts that are proposed is that EcoRII cuts both strands at C↓C in 5′-CCWGG with an axis of symmetry through W, whereas it cuts 5′-CCAG/CTGG once at 5′-C↓CAG. Shortening of the cognate EcoRII sequence from pentamer to tetramer increases the number of cognate loci 5-fold from 8 to 39, with 31 of them predicted to be cut by EcoRII in one strand only (Table I). Finally, cytosine methylation prevents cutting of all 5′-CCWGG and 5′-CCAG sequences (lane 7), whereas purine methylation does not (lane 9). The results of lane 9 demonstrate a dual pattern of dam-methylation that is explained by susceptibility of each plasmid molecule to EcoRII ds cuts at the four 5′-CCWGG loci in one orientation or alternatively at the four 5′-CCWGG loci in the other orientation (the analysis is illustrated in Fig. 2). When one parental strand is methylated at adenine and the other at guanine, semiconservative assortment of parental strands (7Meselson M. Stahl F.W. Proc. Natl. Acad. Sci. U. S. A. 1958; 44: 671-682Crossref PubMed Google Scholar) dictates that one offspring inherits methylated adenine and the other inherits methylated guanine. One concludes from the alternative sensitivity of plasmid populations to EcoRII cuts that one-strand methylation of adenines or of guanines is systematically removed after duplication. The extreme sensitivity of L-DNA and not H-DNA to piperidine (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 9Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 499-560Crossref PubMed Scopus (9015) Google Scholar, 11Johnston B.H. Rich A. Cell. 1985; 42: 713-724Abstract Full Text PDF PubMed Scopus (227) Google Scholar) suggests that methylguanines and not methyladenines are demethylated or replaced in the tetramers after DNA duplication, including those within the pentamers. Methylguanine in L-DNA cannot be identified directly, because the marker deoxy-7-methylguanosine is not available. The analysis illustrated in Fig. 2 identifies ds cuts at all 5′-CCWGG loci except for those designated as 1 and 4 in the map of Fig. 1. A strategy that serendipitously identified the sequence 5′-CCAG and its complement 5′-CTGG for methylation was as follows. In the course of doing in vivo footprinting of the origin (4Maas R. Wang C. Maas W.K. J. Bacteriol. 1997; 179: 3823-3827Crossref PubMed Google Scholar, 12Sasse-Dwight S. Gralla J.D. Methods Enzymol. 1991; 208: 146-168Crossref PubMed Scopus (112) Google Scholar), controls were carried out by piperidine hydrolysis of isolated plasmid to confirm that there was no methylation of purines in the origin region, because the origin has no canonical GATC sequences. The piperidine-treated DNA was used as template in thermally cycled primer extensions with Taq polymerase and a radioactive precursor (4Maas R. Wang C. Maas W.K. J. Bacteriol. 1997; 179: 3823-3827Crossref PubMed Google Scholar). Separation of the extended primers by denaturing PAGE revealed that the assumption of no methylation was incorrect, for there were nick signals at the two tetramers. Such a strategy, using sequencing lanes for location of the nicks (4Maas R. Wang C. Maas W.K. J. Bacteriol. 1997; 179: 3823-3827Crossref PubMed Google Scholar) and plasmid isolated from a dam- host as control should directly identify all As and Gs that become methylated irrespective of recognition by a methylation-sensitive restriction enzyme. Although straightforward, the experiments are beyond the scope of this work. The fragments of lane 9 add up to a 6-kb length, whereas the miniplasmid is 5-kb in size. Amplification of the DNA was eliminated by the complete in-lab sequencing of five plasmid preparations originating from five isolated transformants of a Dam+dcm- host and one transformant of a Dam+Dcm+ host. Both lane 3 and lane 7 demonstrate that when methylation prevents restriction, EcoRII shifts the equilibrium between normal density DNA, named H in my previous publication (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), and L-DNA toward L. The transition to L facilitates replication (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), suggesting that methylation and methylation restriction systems provide an advantage at the time of DNA transfer. Could the absence of optimal replication explain the apparent protection from infecting DNA in the phenomenon of restriction of phage infection? ExoIII as an Enzyme That Alters DNA Helical Density—The topological transition of L-DNA to H is known to occur at the end of each cycle of replication. A reaction analogous to the transition has been demonstrated in vitro by treatment of H-DNA with one of the type I topoisomerases and subsequent treatment of the low linking number reaction product with a type II topoisomerase. Having obtained evidence for the loss of guanine methylation from DNA raises the possibility that in vivo specific demethylation of L-DNA is essential for the transition. Hosts containing L-RepFIC have absolute sensitivity to hydrogen peroxide, possibly as a result of the presence of methylguanine in L-DNA, and do not grow in rich media containing as little as 1 mm hydrogen peroxide. One expects that cells unable to shift the helical density of their DNA from L to H as a result of the presence of methylguanine in their DNA have the same phenotype. ExoIII mutants are exquisitely sensitive to hydrogen peroxide (13Imlay J.A. Linn S. J. Bacteriol. 1986; 166: 519-527Crossref PubMed Scopus (261) Google Scholar). A 3′ → 5′ exonuclease activity of ExoIII on ds DNA oligonucleotides has given the enzyme its name. ExoIII possesses multiple hydrolytic activities toward the sugar phosphate backbone, all determined with in vitro generated substrates and described in Ref. 8Kornberg A. Baker T. DNA Replication. 2nd Ed. W. H. Freeman and Company, New York, NY1992Google Scholar. ExoIII removes from the 3′-end of duplex polymers up to three mispaired nucleotides. In nicked substrates the nick can be enlarged to a gap. In addition ExoIII makes incisions at apurinic and apyrimidinic sites in vitro (14Little J.W. J. Biol. Chem. 1967; 242: 679-686Abstract Full Text PDF PubMed Google Scholar). These activities do not apply to covalently closed circular genetic elements, and they do not explain the sensitivity of ExoIII mutants to hydrogen peroxide. It has been published, however, that ExoIII may generate a particular DNA conformation required to induce heat shock response, defining an ExoIII topological activity in vivo (15Paek K.H. Walker G.C. J. Bacteriol. 1986; 165: 763-770Crossref PubMed Google Scholar). The action of commercially available ExoIII was explored in this work with in vivo isolated plasmid preparations containing mixtures of H- and L-DNA and one preparation containing L-DNA only. Three preparations originated from a Dam+ host and three from a dam- host. The results are shown in Fig. 3. Comparison of each untreated sample (odd-numbered lanes) with the adjacent ExoIII-treated sample shows that ExoIII lowers the electrophoretic mobility of L-DNA and L-DNA only to the lowest in the gel (labeled LL). Dam-methylation does not affect the reaction. The above results were obtained in 0.66 mm magnesium (Mg2+) buffer. To test whether more Mg2+ was required for an additional reaction, the following experiment, shown in Fig. 4, was done. LL-DNA was prepared by ExoIII treatment of L- and H-DNA mixtures, because the relative insolubility of L-DNA (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) hampers its purification by extraction from gels. The pure material seen in Fig. 3 (lane 3) can only be obtained from cells where the L-conformation has been imprinted, i.e. only from Dam+ cells. The reaction tubes were maintained at 70 °C for 20 min (Fig. 4, step I). Mg2+ (lanes 3 and 7) or Mg2+ + ExoIII (lanes 4 and 8, indicated by arrows) was added in a second step. The addition of Mg2+ regardless of the addition of fresh ExoIII resulted in a clear transition of LL-DNA, but only when the DNA was isolated from a dam- host (lanes 7 and 8). ExoIII does not degrade intact circular DNA (8Kornberg A. Baker T. DNA Replication. 2nd Ed. W. H. Freeman and Company, New York, NY1992Google Scholar). L-DNA is circular and supercoiled (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The clarity of the lanes in the position where linear molecules are expected and below the H band, as well as the lack of increase in ethidium-bromide staining material at the bottom of the gel (not shown), confirm that there was no degradation by ExoIII. The transition of LL-DNA in lanes 7 and 8 was most likely to H-DNA, an interpretation that was easier to see with the naked (and protected) eye directly on the UV transilluminator. The purpose of the treatment at 70 °C was inactivation of ExoIII. The L to H transition proceeded in the absence of further addition of ExoIII, suggesting either that the transition was spontaneous or that ExoIII is not inactivated once bound to DNA. The latter seems more likely. When all possible purines were methylated as they normally are in L-DNA, the addition of Mg2+ resulted in diffuse bands of low helical density (lanes 3 and 4). The diffuse bands of LL-DNA could indicate Mg2+ promoted nicking as is seen with topoisomerases and poor staining caused by inefficient intercalation of ethidium bromide in the nicked structure. Postreplicative specific loss of guanine methylation and the in vitro topological activity of ExoIII can be integrated in a model that is considered in the following “Discussion.” This work has provided evidence for methylation in both strands of the tetramer sequence motif 5′-CCWG at guanine, adenine, and cytosine in E. coli. The sequence, which contains three G-C pairs, is present 39× in 3000 base pairs. Seventeen of the tetramers are located in the 48% G-C origin sequence. Full methylation optimizes initiation of the duplication process (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) by stabilizing the prereplicative L-DNA conformation. The activated state of replication cannot be removed until methylguanines are replaced or demethylated. L-DNA that is hemimethylated at de novo purines appears to act as a checkpoint for reversal of specific programmed methylation. As will be discussed, de novo methylated cytosines may be removed as well by an analogous pathway. ExoIII is here proposed to play a pivotal role in the reversal of DNA modifications that alter DNA function. ExoIII conditional mutants exhibit filamentous growth like E. coli where maintenance of the RepFIC plasmid has been imprinted as L, suggesting that sufficient ExoIII is essential for the L to H transition. This in turn suggests that LL-DNA is an activated intermediate for the alteration or removal of DNA units that interfere with the B-helix. Once interference is removed, the ExoIII reaction is completed. It would be interesting to determine whether combining the ExoIII mutation known as xth with a dam mutation relieves both the filamentous growth phenotype and sensitivity to hydrogen peroxide. Relief of filamentous growth would signify that in the absence of a requirement for demethylation of methylguanine, a type II topoisomerase could promote the L to H transition. Relief of sensitivity to hydrogen peroxide would suggest that the sensitivity is indeed due to the presence of methylguanine in DNA. Just as mammals methylate cytosine de novo during development (16Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4574) Google Scholar), E. coli methylate adenine and guanine de novo during replication, resulting in changing patterns of methylation during each replication cycle and leading to the conclusion that one common feature of de novo DNA methylation is the optimization of DNA replication. The properties of the two major methyltransferases in E. coli as well as those of cognate restriction enzymes used in sequence identification are summarized in Table I. Interestingly, the same motif that is methylated de novo by dam-Mtase is methylated by dcm-Mtase, the latter not found in all E. coli strains. In vivo dam-methylated L-DNA appears to be identical to DNA alkylated in vitro (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The major product of in vitro ds DNA alkylation is N7-methylguanine (9Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 499-560Crossref PubMed Scopus (9015) Google Scholar, 11Johnston B.H. Rich A. Cell. 1985; 42: 713-724Abstract Full Text PDF PubMed Scopus (227) Google Scholar). The major methylated purine product by a large factor after exposure of E. coli to alkylating agents is also N7-methylguanine (17Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995: 110Google Scholar). The indications thus are that methylguanine in L-DNA is modified at N7. It is likely that methylguanines are demethylated rather than excised for the following reason. E. coli adapt rapidly when exposed to mutagenic methylating agents (18Samson L. Cairns J. Nature. 1977; 267: 281-283Crossref PubMed Scopus (569) Google Scholar). Adaptation is via the inducible protein Ada, specific for removing methyl adducts from the O6 position of methylguanine (18Samson L. Cairns J. Nature. 1977; 267: 281-283Crossref PubMed Scopus (569) Google Scholar, 19Sassanfar M. Dosanjh M.K. Essigmann J.M. Samson L. J. Biol. Chem. 1991; 266: 2767-2771Abstract Full Text PDF PubMed Google Scholar). The O6 position is vital to hydrogen bonding (20Watson J.D. Crick F.H.C. Nature. 1953; 171: 737-738Crossref PubMed Scopus (8554) Google Scholar), and not surprisingly a system evolved for removing the modification, certain to be mutagenic in view of improper O6-methyl pairing opposite the 4-amino group of cytosine. The question arises why does E. coli demethylate a minor product of methylation when N7-methylguanine is the major modification product and is in fact removed. One explanation, the simplest, is that O6-methylguanine is a functional intermediate marked for demethylation. The methyl group of O6-methylguanine is transferred to cysteine-S of the Ada protein itself (21Schendel P.F. Defais M. Jeggo P. Samson L. Cairns J. J. Bacteriol. 1978; 135: 466-475Crossref PubMed Google Scholar). Methylated Ada protein is a transcriptional activator of the ada operon (22Demple B. Nucleic Acids Res. 1986; 14: 5575-5589Crossref PubMed Scopus (16) Google Scholar). The Ada enzyme is also known as O6-methylguanine-DNA methyltransferase (23Demple B. Jacobsson A. Olsson M. Robins P. Lindahl T. J. Biol. Chem. 1982; 257: 13776-13780Abstract Full Text PDF PubMed Google Scholar). The ada operon transcribes one additional protein that has been named AlkB (24Kataoka H. Yamamoto Y. Sekiguchi M. J. Bacteriol. 1983; 153: 1301-1307Crossref PubMed Google Scholar, 25Kataoka H. Sekiguchi M. Mol. Gen. Genet. 1985; 198: 263-269Crossref PubMed Scopus (51) Google Scholar). The in vitro activity of AlkB is unknown. In vivo absence of AlkB sensitizes the cell to killing by the methylating agent MMS (24Kataoka H. Yamamoto Y. Sekiguchi M. J. Bacteriol. 1983; 153: 1301-1307Crossref PubMed Google Scholar). Could AlkB take part in the methyl group transfer from N7 to O6 and thus be part of the guanine demethylation reaction? There is an additional protein in E. coli that has the potential to demethylate O6-methylguanine. It is not inducible. Ada and the noninducible DNA methyltransferase remove methyl groups from both O6-methylguanine and O4-thymine, neither one of which is a major product of in vivo alkylation reactions. Ada protein displays greater efficiency in the demethylation of O6-meG and the noninducible enzyme in the demethylation of O4-meT. The noninducible protein is conserved in yeast and man, where it has the same preferential activity toward O4-meT (19Sassanfar M. Dosanjh M.K. Essigmann J.M. Samson L. J. Biol. Chem. 1991; 266: 2767-2771Abstract Full Text PDF PubMed Google Scholar). The similarities of the two DNA methyltransferases and the conservation of O4-meT-DNA methyltransferase through the evolutionary ladder suggest a normal biological function for both DNA methyltransferases that is advantageous but does not necessarily reflect a response to mutagen exposure. 7-Methylguanine remains in the DNA helical wall of Z-DNA (26Moller A. Nordheim A. Nichols S.R. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4777-4781Crossref PubMed Scopus (65) Google Scholar) and presumably in the helical wall of L-DNA (1Maas R. Cell. 2001; 105: 945-955Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Exposure in the wall of the helix could be one recognition basis for base alterations. Methylcytosine on the other hand is frequently found in supercoiled B-DNA. Thus, when methylcytosine is removed it is recognized on a different basis. A strategy used by the scientist in the laboratory to identify methylcytosine in DNA is to convert it to uracil (27Grigg G. Clark S. Bioessays. 1994; 16: 431-436Crossref PubMed Scopus (34) Google Scholar, 28Clark S.J. Harrison J. Paul C.L. Frommer M. Nucleic Acids Res. 1994; 22: 2990-2997Crossref PubMed Scopus (1627) Google Scholar). The strategy appears to have its biological counterpart, and furthermore ExoIII is involved in the removal of uracil from DNA. Methylcytosine is altered in vivo by hydrolytic deamination to thymine. When dcm-methyltransferase is overproduced in E. coli, G-C → T-A transitions are increased (29Bandaru B. Gopal J. Bhagwat A.S. J. Biol. Chem. 1996; 271: 7851-7859Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), suggesting saturation of a system that normally corrects the meC → T transitions. Unambiguous correction of the mismatch at the modified base further suggests that transitions and their reversion are part of DNA metabolism. In fact, the enzyme responsible for meC → T transitions has been described in E. coli (30Yebra M.J. Bhagwat A.S. Biochemistry. 1995; 34: 14752-14757Crossref PubMed Scopus (78) Google Scholar). dcm-methyltransferase deaminates its own product 5-methylcytosine to thymine in the absence of methyl donor S-adenosylmethionine (30Yebra M.J. Bhagwat A.S. Biochemistry. 1995; 34: 14752-14757Crossref PubMed Scopus (78) Google Scholar, 31Sharath A.N. Weinhold E. Bhagwat A.S. Biochemistry. 2000; 39: 14611-14616Crossref PubMed Scopus (26) Google Scholar). Methyl transfer within T in G-T mismatches from C5 to O4 could provide an intermediate analogous to O-methylguanine, giving rise to uracil when demethylated. Uracil opposite G in DNA is corrected by an active dedicated glycosylase, conserved from bacteria to man, and the correction is dependent on ExoIII in E. coli and presumably on the ExoIII homolog HAP1 in man. Thus, the above series of reactions provides a fool-proof system for the unambiguous removal of meC. The methylcytosine removal process ends in mutation, however, when the T-to-U transition followed by U excision does not take place. It has been suggested that DNA “damage” results from metabolites present in the cell (32Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4378) Google Scholar, 33Mackay W.J. Han S. Samson L.D. J. Bacteriol. 1994; 176: 3224-3230Crossref PubMed Google Scholar). This work provides evidence that methylation of guanine, a modification that optimizes replication, is a normal cellular process that is reversed, deactivating replication. Methylated bases are in fact detected in the DNA of cells that have not been exposed to exogenous mutagens (34Park J.W. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7467-7470Crossref PubMed Scopus (103) Google Scholar, 35Tan B.H. Bencsath F.A. Gaubatz J.W. Mutat. Res. 1990; 237: 229-238Crossref PubMed Scopus (36) Google Scholar). Under the circumstances DNA “lesions” (36Gonzalgo M.L. Jones P.A. Mutat. Res. 1997; 386: 107-118Crossref PubMed Scopus (184) Google Scholar) can be explained by the failure to revert programmed DNA alterations. An understanding of mechanisms that regulate DNA function by reversible alterations of the DNA structure or the code itself could lead to new therapeutic approaches for reducing cancer risk, because insufficiencies in DNA metabolism as well as mutagenic agents may result in damaging mutagenesis. The technical assistance of Frank Alessandro was invaluable. I have appreciated discussions with many colleagues, particularly Werner K. Maas. I am indebted to Ashok Bhagwat for the generous gift of high quality EcoRII. I am also grateful for the generosity of my chairman, Claudio Basilico." @default.
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