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Interactions in Escherichia Coli NAT STERNBERG Central Research and Development Department Experimental Station, E 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.
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