JOURNAL OF BACTERIOLOGY, OCt. 1985, p. 490(493 Vol. 164, No. 1 0021-9193/85/100490-04$02.00/0 Copyright © 1985, American Society for Microbiology Evidence that Adenine Methylation Influences DNA-Protein Interactions in Escherichia coli NAT STERNBERG Central Research and Development Department Experimental Station, E. l. Du Pont de Nemours and Co., Inc., Wilmington, Delaware 19898

In this review, most of the information presented will be functional recA gene. K. R. Peterson, K. F. Wertman, derived from studies with Escherichia coli K-12 (E. coli) and D. W. Mount, and M. G. Marinus have recently found that its related bacteriophages, simply because more is known the genes of the SOS regulon could be induced (1.7- to about methylation in these organisms than in any other. The 6-fold) by introducing a dam mutation into E. coli. The two methylated bases that have been detected in E. coli are affected genes include recA, lexA, uvrA, uvrB, sulA, dinD, 6-methyladenine (6-meAde) and 5-methylcytosine (7, 18). and dinF. The only SOS gene not effected is uvrD. Except Since little is known about the biological function of 6- for lexA and uvrB, none of the genes are fully induced. In methylcytosine, I will deal exclusively here with the studies essence, the cell adjusts the basal level of the SOS response on 6-meAde. to that needed without fully inducing the regulon. In this 6-meAde is the product of a reaction catalyzed by a case, the control of gene expression is probably an indirect methylase coded for by the bacterial dam gene (21). In the effect of the methylation defect. Presumably, the absence of absence of that methylase, less than 1% of the 6-meAde methylation produces a cascade of events that includes normally found in DNA remains (2, 21, 22). The specific random mismatch repair, increased single-stranded breaks, sequence recognized by the methylase is 5'-GATC-3' (4, 9, inactivation of lexA repressor, and SOS induction. More- 14). Dam- mutants exhibit a variety of phenotypes that over, since recA dam, lexA dam, and ruv dam double include increased spontaneous mutability (5, 23), increased mutants are not viable (21, 22), it would appear that the sensitivity to agents such as UV light or 2-aminopurine (22), induction of at least one SOS regulon gene (the ruv gene) is hyperrecombination (20), and increased transposon- essential for the viability of dam mutants. mediated rearrangement of DNA (13; D. Roberts and N. Transposon function. The most extensively studied case of Kleckner, personal communication; J. Yin and W. Dam-mediated gene regulation involves the transposon Reznikoff, personal communication). A lack of 6-meAde has TnOO. TnlO-mediated transposition is elevated 10-fold, and also been correlated with either an increase or decrease in TnIO-mediated deletion or inversion is elevated 100- to the expression of genes (8, 12, 19; N. Sternberg, unpublished 250-fold in a Dam- cell (13; D. Roberts and N. Kleckner, data), a decreased replication of replicons (24, 30), and a personal communication). Other transposons which show a decreased packaging of viral DNAs (31; N. Stemnberg, 5- to 10-fold elevation in transposition in Dam- cells are unpublished data). Tn9O3 (13), Tn5 (13; J. Yin and W. Reznikoff, personal In Dam- cells, both strands of the DNA are unmethylated. communication), and IS50 (J. Yin and W. Reznikoff, per- In normal cells, however, this situation rarely exists, be- sonal communication). The Dam methylase appears to op- cause the old template strand in a replicating DNA duplex is erate at two levels to repress transposon function (D. already methylated, and the newly synthesized DNA strand Roberts, N. Kleckner, J. Yin, and W. Reznikoff, personal is methylated soon after its replication (16, 17, 32). Thus, communication). First, methylation of critical Dam sites in whatever effects are attributed to the absence of 6-meAde in the -10 region of the promoter for the transposase gene DNA, they must be manifested during the short period of reduces transcription of the gene and also the amount of time when the newly synthesized DNA strand is transiently transposase made, by about 5- to 10-fold. Second, methyla- unmethylated, namely, when the DNA is hemimethylated. A tion of Dam sites at the inside ends of insertion elements general idea that appears to be emerging from studies on the (ISIO for TnWO and IS50 for TnS) decreases the ability of the biological role of 6-meAde is that gene expression can be transposase to utilize those ends for transposition or rear- coupled to DNA replication and cell division if the gene is rangement. The more pronounced effect of the Dam- phe- transcribed poorly from fully methylated DNA and effi- notype on TnJO-mediated inversion or deletion, compared ciently from hemimethylated DNA. Examples of biological with transposition, reflects the preferential use of the inside systems in which gene expression and protein function are end of the insertion element for the former class of rear- affected by adenine methylation are described below. I will rangement. not discuss in this review the extensive literature that deals The promoter sequences of the TnJO and Tn5 transposase with the role of Dam methylation in the repair of mismatched genes are shown in Fig. 1A (6; J. Yin and W. Reznikoff, DNA. Reviews that discuss this subject have recently ap- personal communication). Both promoters contain a 6- peared or are about to appear in the literature (18; M. meAde nucleotide just 5' to the -10 region. TnS also has a Marinus, personal communication). second 6-meAde within the -10 region. The importance of The SOS response. When the DNA metabolism ofE. coli is these nucleotides is indicated by a TnJO mutation that perturbed, the cells respond by inducing a set of genes, converts the G residue upstream of the -10 region to an A collectively referred to as the SOS regulon, whose function (Fig. 1A). This mutation (DR33) eliminates the Dam is necessary for cell survival under those stressful conditions methylation site and renders transcription mediated by this (15, 33). An important step in that induction process is the promoter, both in vivo and in vitro, insensitive to Dam inactivation of a repressor of the SOS regulon that is methylation (D. Roberts, N. Kleckner, B. Hoopes, and W. encoded by the lexA gene. lexA inactivation requires a McClure, personal communication). The sequences of the 490 VOL. 164, 1985 MINIREVIEW 491

A. Promoters <---- -35.-><----- spacer .--X-10> Consensus CTTGACAAT ---- - 13-18 bp------TATAAT Transposase-TnlO (Pin) GTGGATACA------13 bp -- GATCAAAT Pin mutant DR33 A Transposase-Tn5 CGGAACCTT------13 bp------GATCTGATC TrpR ACTGATCCG 16 b . TATCGT sulA GTTGATCTT------15 bp------TACTGT Pri (Picre) CTTGATCATTTGATCAA------11 bp--- TAAAAT

B. IS inside ends .---ISelement------> 'S10 ATGAGGGGATCTCTCAG IS10 mutant DR7 T IS50 AGATCTGATCAAGAGACAG FIG. 1. The sequences of Dam-sensitive promoters and inside termini of insertion elements. The consensus promoter sequence is from Hawley and McClure (11). The sequence of the Tn5 transposase promoter shown is not that originally recognized as that of the promoter for the transposase (11; M. Krebs, J. Yin, and W. Reznikoff, personal communication). Only the altered bases in the two TnJO mutants, DR33 and DR7, are shown (D. Roberts and N. Kleckner, personal communication ). The Dam methylation sites are underlined. Pin, Promoter for the TnlO transposase gene; Prl, promoter for the cre gene. inside ends of ISIO and IS50 are shown in Fig. 1B (3, 6). Both the TnJ0 transposase promoter is weak and that transposi- sequences contain Dam sites in the regions that are thought tion can be increased by replacing it with a stronger pro- to interact with the transposase. In an experiment in which moter (26). the TnJO transposase was provided by a Dam-insensitive Other Dam-sensitive genes. The following four genes have promoter, transposition of elements utilizing the inside end been identified that also show a sensitivity to Dam methyla- of ISIO was shown to be elevated 6- to 10-fold in a Dam- tion: E. coli trpR (M. Marinus, personal communication), E. host (D. Roberts and N. Kleckner, personal communica- coli sulA (25; S. Gottesman, personal communication), tion). This effect was eliminated by a mutation that converts bacteriophage P1 cre (unpublished data), and bacteriophage the GATC sequence at the inside end of ISIO to GATT (DR7; Mu mom (8, 10, 12, 28). The first three genes contain Dam Fig. 1B). methylation sites in the -35 region of the promoters (Fig. In Dam' cells, the stimulation of transposition produced 1A). Their transcription is elevated two- to sixfold in a Dam- when the DNA is undermethylated most probably occurs host compared with the isogenic Dam' host. For sulA, part only during the short period after the transposon has been of this induction (two- to threefold) is indirect and is due to replicated and before the newly replicated DNA strand has partial SOS induction, but the remainder (two- to threefold) been methylated. If this is true, one might expect to find that is due to a direct effect of methylation at the promoter (S. undermethylation of one of the two DNA strands is more Gottesman and M. Marinus, personal communication). critical than that of the other. These predictions were While the critical experiments have not yet been done, the confirmed in experiments carried out by D. Roberts and N. activation of these three promoters, like that of the Kleckner (personal communication). DNA in which only transposase genes, probably involves the production by one of the two strands is methylated (hemimethylated DNA) replication of hemimethylated promoter DNA. In this re- was generated in vivo by single-stranded Hfr transfer fol- spect, Marinus (personal communication) has argued that lowed by complementary strand synthesis in the recipient the location of the tryptophan operon repressor gene (trpR), cell. Transfer of two ISIO elements inserted at the same site, between the bacterial origin of replication and the but in opposite orientations, results in hemimethylated IS1O tryptophan operon, ensures a burst of repressor synthesis elements which are methylated on opposite strands. Trans- before the operon is replicated. The P1 Cre protein is a position from the newly transferred and replicated DNA was site-specific recombinase (31) that acts to cyclize the linear measured. A 10-fold difference was observed in the Dam' P1 viral DNA after its injection into E. coli. Cre must act recipient for the two orientations. This difference was en- quickly before the DNA is degraded. While the cre promoter hanced only an additional twofold in the Dam- recipient. is quite weak, it is expressed three to four times more These results argue that most of the transposition events efficiently in a Dam- strain than in a Dam' strain (N. occurred during the hemimethylated state created by the Sternberg, unpublished data). This might be biologically DNA transfer process. This conclusion is supported by the relevant if the cre gene were rapidly replicated after viral observation that a similar orientation difference was not injection. The resulting hemimethylated or perhaps even observed with an isogenic mutant of ISIO that lacked the unmethylated state of the cre promoter would result in an GATC sites (TnJO DR33 and DR7). increased transcription of the gene and a sufficient level of The single-strand transfer experiment supports the con- recombinase for the cyclization reaction. tention that transposition is modulated by the natural loss of The Mu mom gene encodes a DNA modification function, full methylation after replication of the transposon. After the expression of which requires the host Dam function. replication, there will be a short interval in which the two Hattman (8) has shown that this regulation operates at the hemimethylated copies of the transposon exist. One of these transcription level since mom transcripts are reduced 20-fold will be activated for transposition until methylation occurs, in a Dam- host. The proposed mom promoter has no Dam at which point transposition will return to a low level. For methylation sites but is just downstream from a region that this model to be valid, the transposase or its ability to act contains a cluster of three Dam sites (10). Removal of some must be limiting. This is, in fact, the case. It is known that of these Dam sites eliminates the dependency on Dam 492 MINIREVIEW J. BACTERIOL. function (10, 12). Based on these and other observations, a DNA (more than a P1 monomer) has been encapsidated. The model for the Dam regulation of mom has been proposed (10) DNA is then cut again, and the next headful initiates at the in which mom is repressed by a protein that binds to the point where the first one terminated. An essential feature of unmethylated mom promoter region but not to the methyl- such a processive headful packaging mechanism is that no ated promoter. It is argued that methylation stabilizes or more than one or, at most, a few pac sites per concatemer induces a DNA conformation (12, 28) that prevents access of should be cleaved. If each pac site were cut at the same time, the protein to the site. However, this model appears to be the concatemer would be converted to monomer-sized ruled out by the demonstration (A. Seiler and R. Kahmann, pieces, and packaging would not be able to generate DNA personal communication) that mutations in the mom regula- with homologous sequences at both ends. Those homolo- tory region which do not effect the proposed DNA confor- gous ends are necessary for cyclizing the viral DNA after its mation make mom expression independent of dam function. injection into a bacterial host. An alternative hypothesis is suggested by the observation The pac site has been localized to a 150- to 160-base-pair (A. Seiler and R. Kahmann, personal communication) that a segment of DNA that contains seven Dam methylation sites, mutH mutation can suppress the inhibition of mom expres- three at one end of the pac site and four at the other (N. sion in a dam- host. Preliminary experiments (K. Welsh, Sternberg, unpublished data). Deletion of either of these two A.-L. Lu, and P. Modrich, personal communication) suggest clusters of Dam sites inactivates pac. Moreover, unmethyl- that the MutH protein cleaves the unmethylated DNA strand ated pac-containing DNA is insensitive to cleavage in vivo at Dam sites in hemimethylated DNA, but not in methylated until it is methylated. J. Coulby and N. Sternberg (unpub- DNA. Taken together, these results point to a model in lished data) have recently shown that P1 encodes its own which the primary cause of the inhibition of mom expression Dam-like methylase. Based on these observations, a model in a Dam- host is the interaction between unmethylated for the regulation of pac cleavage has been proposed in DNA upstream of the mom gene and MutH protein. which a limiting step in the process is the activation of pac In addition to affecting gene expression, Dam methylation by the methylation of unmethylated Dam sites. During the appears to affect other protein-DNA interactions as well. early stages of the P1 vegetative cycle, pac is largely Examples of two such interactions, DNA replication and unmethylated and therefore inactive, because replication of DNA packaging, are discussed below. the P1 DNA is occurring rapidly and the host methylation Replication initiation. The E. coli origin of replication, system cannot fully methylate it. Later in the infection cycle, oriC, has been localized to a region of 250 base pairs that some of the pac sites became methylated, due to the contains 12 to 14 Dam methylation sites (27). Evidence for synthesis and action of the virus-encoded methylase. As the importance of these sites in origin function comes from soon as that occurs, pac becomes a substrate for the the following three sources. (i) Eight of the oriC Dam sites initiation of packaging, and packaging itself then blocks are conserved among origins from five gram-negative bacte- further utilization of the other pac sites. By requiring multi- ria (34). (ii) Plasmids that use oriC for initiation of replication ple methylation steps to activate pac, the system ensures transform Dam- strains either not at all or very inefficiently. that two pac sites on the same concatemer will rarely Moreover, oriC DNA isolated from Dam- cells functions become activated at the same time. two- to fourfold less well in an oriC-specific in vitro initiation The data summarized in this review clearly demonstrates system than does oriC DNA from Dam' cells. Methylation that DNA methylation at 5'-GATC-3' sites is important in of Dam- DNA in vitro restores normal template activity regulating gene expression and DNA processing in E. coli (31). (iii) Increased levels of Dam methylase in cells, due to and in its bacteriophages. The fact that the methyl group at a dam' gene on a high-copy-number plasmid, reduce the the N6 position of adenine projects into the major groove of spacing between replication initiation events (24). The latter DNA suggests that methylation may play a significant role in result supports the contention that the spacing of replication the recognition of Dam sites by various proteins. In partic- forks is regulated by the time required for the methylation of ular, it may affect specific interactions between RNA poly- the newly replicated hemimethylated DNA. A model to merase and promoters containing Dam sites. Those interac- account for this, based on the stabilization of DNA tions need to be studied in greater detail. Also of particular intrastrand secondary structure at oriC by Dam methylation, importance will be studies that deal with the function of has been proposed (30). those Dam methylases, such as that encoded by bacterio- Smith et al. (30) have shown that the Dam sites in oriC phage T4 (29), whose biological role has not yet been DNA isolated from Dam- mutants exhibit some sensitivity identified. The next few years promise to contain some to digestion by DpnI, a restriction endonuclease specific for exciting additions to the methylase-gene function story. methylated GATC sites (4). They conclude that these mutant strains (one of which contains a Tn9 insertion in the dam gene) must retain an activity that can methylate at least some ACKNOWLEDGMENTS oriC Dam sites. The source of that methylase still needs to be determined. P. Janczyk and D. Smith (personal commu- I thank D. Roberts, N. Kleckner, B. Hoopes, W. McClure, W. Reznikoff, R. Kahmann, D. Smith, W. Messer, M. Marinus, P. nication) have recently uncovered another regulatory circuit Modrich, and S. Hattman for communicating information before involving the dam gene. Some dnaA(Ts) mutants at 38°C publication. I also thank R. Hoess for critical reading of the exhibit 5 to 10 times more transcription from the dam gene manuscript. promoter than does the isogenic dnaA+ cell. This result suggests that the DnaA protein (an essential oriC initiation protein) may also be a repressor of dam. ADDENDUM IN PROOF The regulation of P1 pac cleavage. The packaging of bacteriophage P1 DNA into a viral capsid is initiated by the Robert Braun (personal communication) has recently cleavage of the DNA at a unique site (pac) on a concatemer shown that Dam methylation regulates the transcription ofthe that consists of repeating P1 monomers (1, 31). Packaging E. coli dnaA gene. In a Dam- strain, transcription of that gene proceeds in one direction from the cut end until a headful of is reduced twofold relative to a Dam' strain. VOL. 164, 1985 MINIREVIEW 493

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