JOURNAL OF BACTERIOLOGY, Aug. 1990, p. 4214-4221 0021-9193/90/084214-08$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 172, No. 8
A Gene Required for Very Short Patch Repair in Escherichia coli Is Adjacent to the DNA Cytosine Methylase Gene ANJUM SOHAIL,"2 MARGARET LIEB,3 MUBASHER DAR,4
AND
ASHOK S. BHAGWATl.4*
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 117241; Centre for Advanced Molecular Biology, University of the Punjab, Lahore-20, Pakistan2; University of Southern California School of Medicine, Los Angeles, California 900333; and Department of Chemistry, Wayne State University, Detroit, Michigan 482024 Received 14 March 1990/Accepted 9 May 1990
Deamination of 5-methylcytosine in DNA results in T/G mismatches. If unrepaired, these mistches can lead to C-to-T transition mutations. The very short patch (VSP) repair process in Escherichia coli counteracts the mutagenic process by repairing the mismatches in favor of the G-containing strand. Previosly we have shown that a plasmid containing an l1-kilobase fragment from the E. coli chromosome can complement a chromosomal mutation defective in both cytosine methylation and VSP repair. We have now mapped the regions essential for the two phenotypes. In the process, we have constructed plasmids that complement the chromosomal mutation for methylation, but not for repair, and vice versa. The genes responsible for these phenotypes have been identified by DNA sequence analysis. The gene essential for cytosine methylation, dem, is predicted to code for a 473-amino-acid protein and is not required for VSP repair. It is similar to other DNA cytosine methylases and shares extensive sequence similarity with its isoschizomer, EcoRl methylase. The segment of DNA essential for VSP repair contains a gene that should code for a 156-amino-acid protein. This gene, named vsr, is not essential for DNA methylation. Remarkably, the 5' end of this gene appears to overlap the 3' end of dcm. The two genes appear to be transcribed from a common promoter but are in different translational registers. This gene arrangement may assure that Vsr is produced along with Dcm and may minimize the mutagenic effects of cytosine methylation. Base mismatches can arise in DNA for a variety of These include replication errors and the action of chemical mutagens that react with DNA (for a review, see reference 30). An additional cause for the generation of base mismatches is the inherent instability of cytosine. Under physiological conditions, cytosine deaminates to uracil at appreciable rates (24). The resulting uracil/guanine mismatches are repaired by a process which, in the first step, involves the removal of uracil (23). 5-Methylcytosine is more unstable than cytosine (10, 24), and its deamination results in the creation of thymine/guanine (T/G) mismatches. If not correctly repaired prior to replication, C-to-T transitions occur. Cytosine methylation sites in DNA have been found to be "hotspots" for such mutations in Escherichia coli (7; M. Lieb, unpublished results). The only DNA cytosine methylase in E. coli K-12, Dcm, transfers a methyl group to position 5 of the internal cytosine in the sequence 5'-CCWGG-3', where W is A or T (29). E. coli also contains a base mismatch correction process that corrects T/G mismatches in sequence contexts such as Cl AGG/GjTCC and CITGG/GGACC to C/G (21). This process, called very short patch (VSP) repair, reduces the mutagenic effect of cytosine methylation (M. Lieb, unpublished results). It is likely that this process arose in E. coli to reduce the mutagenicity of 5-methylcytosine. Marinus and Morris used a chemical mutagen to obtain a series of E. coli mutations, dcm-1 through dcm-11, that were defective in the methylation of cytosines in DNA (28). One of these mutations, dcm-6, was later shown to lead to the loss of VSP repair in the cell as well (14, 20, 40). This led to the conclusion that Dcm was required for VSP repair and might be a bifunctional protein. We recently showed that a plasmid containing an 11-kilobase fragment of chromosomal
DNA from wild-type E. coli complements both the methylation and repair defects of dcm-6 (22). This is consistent with the suggestion that Dcm plays a direct role in VSP repair. In that study, we also tested the gene for another methylase, EcoRII, for its ability to complement dcm-6 for its repair defect. EcoRII methylase is part of the EcoRII restrictionmodification system and has sequence specificity identical to that of Dcm (29, 35). Hence, the gene that codes for the EcoRII methylase complements dcm-6 for methylation. Interestingly, it did not restore VSP repair in the cell (22). As a result of these observations, it was concluded that the ability of EcoRII to methylate Dcm/EcoRII sites is not sufficient for that enzyme to have a role in VSP repair. We report here that this is true of Dcm as well. Contrary to previous reports, dcm plays no direct role in VSP repair. Instead, a gene adjacent to it is required for this process. In turn, this gene appears to play no role in DNA methylation. (A preliminary report of this work was published [4] as the summary of a paper presented at the New England BioLabs Workshop on Biological DNA Modifications, Gloucester, Mass., 20 to 23 May 1988.)
reasons.
*
MATERIALS AND METHODS
Bacterial strains. GM1 [F- thr-l ara-14 leuB6 A(gptproA)62 lac Yl tsx-33 supE44 galK2 hisG4 rflhbD mgl-S1 rpsL31 kdgKSJ xyl-S mtl-l metBI thi-1]; its derivatives carrying various dcm alleles; GM30 (thr-J hisG4 leuB6 rpsL ara-14 supE44 lacYl tonA31 tsx-78 galK2 galE2 xylS thi-l mtl-i) and GM31 (i.e., GM30 dcm-6) were kindly provided by M. G. Marinus (University of Massachusetts School of Medicine, Amherst, Mass.). RP4182 [A(supD-dcm-fla) trp gal rpsL] was provided by J. S. Parkinson (University of Utah, Salt Lake City, Utah). Assay for methylation of Dcm sites. The ability of a bacterial strain to methylate Dcm/EcoRII sites was assessed
Corresponding author. 4214
GENE REQUIRED FOR VSP REPAIR IN E. COLI
VOL. 172, 1990
in two different ways. Plasmid DNAs from the strains
TABLE 1. VSP repair phenotypes of dcm mutations
were
routinely digested with EcoRII endonuclease (Bethesda Research Laboratory, Rockville, Md.), and the result was analyzed by electrophoresis on an agarose gel. Methylation of the Dcm/EcoRII sites results in the protection of the plasmid DNA against cleavage. Quantitation of the methylase activity was done for selected strains by determining the number of 3H atoms transferred from [3H]adenosyl-L-methionine (Dupont, NEN Research Products, Boston, Mass.) to chromosomal DNA from RP4182 as described before (5). Assay for VSP repair. The percentage of cI+ progeny among N+ O+ recombinants resulting from a cross of bacteriophage mutants Nam53 cIam6 O+ and N+ cI CP7 Oam29 was used to quantitate VSP repair as described before (21). Mutation am6 changes a CCAGG sequence to CTAGG; mutation CP7 is a T-to-A transversion located 20 base pairs (bp) to the left of am6 on the X map. Briefly, bacteria were infected with a 1:1 mixture of the two mutants to give a total multiplicity of infection of 10. After a 15-min adsorption period at 37°C, broth was added and incubation
continued for a total of 100 min, followed by the addition of CHCl3 and vortexing. Recombinants that are N+ O+ were selected by plating the phage progeny on a lawn of Supo bacteria; N+ O+ phage that are also cI+ form turbid plaques, while phage that carry the am6 or the CP7 mutation in cI form clear plaques. N+ O+ recombinants that are also cI+ require three crossover events and are expected to be very rare. However, during the recombinational events leading to N+ O+ phage, heteroduplex regions span the intervening cI gene, resulting in am6/+ (T/G) mismatches. Correction of am6 (T) to (C) results in excess N+ cI+ O recombinants. Therefore, the fraction of cI+ among the recombinant progeny phage is a measure of VSP repair in the host. Recombinant DNA work and DNA sequencing. Standard techniques were used in the construction of Bal3l-mediated deletions, deletion of restriction fragments, and construction of M13 phage clones (26). Inserts in the M13 clones were sequenced by using appropriate primers by the technique of Sanger et al. (34). was
RESULTS VSP repair and Dcm phenotypes can be unlinked. Three research groups have independently reported that E. coli K-12 cells carrying the dcm-6 mutation are deficient in VSP repair (14, 20, 40). dcm-6 is 1 of the 11 independent alleles of dcm isolated by Marinus and Morris by chemical mutagenesis of the strain GM1 (28). The nature of the mutation(s) is not known for any of the alleles. Three of the dcm mutant alleles have been lost; we have tested five of the remaining alleles for their effect on VSP repair. The results are summarized in Table 1. When a bacteriophage X cross diagnostic for VSP repair was done in GM1, the percentage of cI+ recombinants obtained was about 1.8% (Table 1). This number is similar to that obtained in another dcm' strain, GM30 (Table 1) (20). In GM31, the dcm-6 derivative of GM30, this number was sixfold lower (Table 1) (20), indicating a lower level of VSP repair. GM1 derivatives containing the alleles dcm-9 or dcm-10 showed a similarly low level of repair activity (Table 1). These mutations caused 10- and 12-fold reductions, respectively, in the Dcm activity in the cell (28). In contrast, GM1 derivatives carrying the alleles dcm4 or dcm-7 showed the same level of repair activity as GM1 and hence are VSP repair proficient (Table 1). Surprisingly, the derivative with the allele dcm-J showed slightly higher VSP repair activity
4215
Strain
GM30 GM31 GM1 GM5 GM8 GM11 GM13 GM14 RP4182
Allele
dcm' dcm-6
dcm' dcm-1 dcm4 dcm-7 dcm-9 dcm-1O Adcm
% of cI+ recombinantsa
2.2 0.36 1.8 6.0 2.0 2.5 0.32 0.49 0.08
± ± ± ±
+
± ± ± ±
0.6 0.15 0.3 1.9 0.5 0.4 0.19 0.07 0.03
a Among N+ O+ progeny (see Materials and Methods). Each value is an average of data from three or more experiments.
than GM1. Mutations dcm-J, dcm4, and dcm-7 cause 6-, 500-, and 2-fold reductions, respectively, in the Dcm activity in the cell (28). It is clear that there is no direct correlation between the Dcm activity and VSP repair activity in these strains. From these data we concluded that, although Dcm and VSP repair are tightly linked to each other, the two phenotypes can be unlinked. It should also be noted that there may not be a complete loss of VSP repair activity in dcm-6-, dcm-9-, or dcm10-containing strains. RP4182 is a strain of E. coli K-12 that carries a large deletion at about 43' on the chromosome and is missing several genes including dcm (2, 5, 17). It consistently produced three- to fourfold fewer cI+ recombinants in a similar cross (Table 1). It is possible that this is a result of the differences in the genetic backgrounds of GM1 and RP4182. It is more likely that there is some residual VSP repair in the strains containing the dcm-6, dcm-9, or dcm-10 alleles, and the reason for the lower numbers of cI+ recombinants in RP4182 is that the deletion in its chromosome eliminates a gene(s) essential for VSP repair. Dcm and VSP repair phenotypes can be separated biochemically. We have shown that a multicopy plasmid, pDCM1, that contains an 11-kilobase-pair DNA fragment from the E. coli chromosome (5), complements the dcm-6 mutation for VSP repair as well as methylation at the Dcm sites (22). Another plasmid, pDCM3, containing an overlapping chromosomal fragment (5), also complements this mutation for methylation and repair (data not shown). pDCM4 was created by deleting a HindlIl fragment within pDCM3 (5). This plasmid also complements the dcm-6 mutation for both the phenotypes, i.e., it has the phenotype methylation-positive VSP repair-positive (M+ Vsr+) (22). Bal3l deletions were generated from three unique sites in pDCM4 (BamHI, StuI, and Sall), and the resulting derivatives were tested for methylation and VSP repair (Fig. 1). Short deletions that removed sequences from the right side of the chromosomal insert had little effect on either phenotype (pDCM22, pDCM23, and pDCM33). Larger leftward deletions from the BamHI site showed two new phenotypes, M+ Vsr- and M- Vsr- (Fig. 1). All constructs that had more than about 2.0 kilobases of DNA removed from the right end were Vsr- (i.e., pDCM8, pDCM9, and pDCM21). One of these plasmids, pDCM21, fully complemented the dcm-6 mutation for methylation. Another, pDCM8, showed a reduced level of methylation activity. DNA prepared from RP4182 containing this plasmid was only partially resistant to the EcoRII endonuclease (data not shown). Deletions from the right end that removed greater than 2.7 kilobase pairs of the insert were defective in methylation as well as in VSP repair (pDCM9; data not shown). The Vsr- phenotype could also be created by the insertion
4216
SOHAIL ET AL.
J. BACTERIOL.
Sail Sau3A/BamHI / Cial Plasmi
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Phenotype
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++
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4 4.3Okbp FIG. 1. Phenotypes of pDCM4 derivatives. Various derivatives of pDCM4 (5) were constructed by Bal3l-mediated deletions, by the deletion of restriction fragments, or by the insertion of a linker. Only those derivatives that were tested for their methylation (M) and VSP repair (VSR) abilities are shown here. Others were used for the sequencing of the DNA segment (see below). Most of the pBR322-derived DNA in these plasmids is not shown. Filled-in rectangles represent DNA derived from the E. coli chromosome; the rest is from pBR322. Plasmids pDCM8, pDCM22, and pDCM23 were generated by Bal3l-mediated deletions from the BamHI site. PDCM9 was generated by ligating a BamHI linker at the StuI site and deleting the smaller BamHI fragment from the plasmid. After the deletion of DNA, BamHI linkers were ligated to the ends and the plasmids were circularized. Plasmid pDCM24 was made by Bal31-mediated deletion from the SalI site. No linker was inserted at the end prior to circularization, and the precise endpoint of deletion is unknown. Hence, the endpoint is indicated by a broken line. Plasmid pDCM33 was made by cloning a SphI-BamHI fragment from pDCM22 into pACYC184. The SphI site used lies within the pBR322-derived DNA in pDCM33. Deletion of AvaI and ClaI fragments from pDCM23 created pDCM28 and pDCM36, respectively. The construction of pDCM35 is described in the text. The ability (or inability) of these plasmids to complement dcm-6 for VSP repair and for methylation of Dcm/EcoRII sites is indicated by a plus or minus sign under the columns labeled VSR and M, respectively. The protection of Dcm/EcoRII sites, as determined by the gel electrophoretic assay, was the criterion used to judge the methylation phenotype. A frequency of cI+ recombinants below 0.5% in standard phage crosses was judged to be indicative of no VSP repair. Cells carrying plasmids indicated to be Vsr+ generally showed a frequency of cI+ recombinants in excess of 4%. 1
of 4 bp at the unique NcoI site in the chromosomal insert (Fig. 1, pDCM35). This was achieved by cutting the plasmid pDCM23 with NcoI and filling in the four nucleotide cohesive tails with DNA polymerase. Success of the fill-in reaction was tested by checking the self-ligated molecules for a new NslI site. This frameshift mutation did not affect the methylation ability of the plasmid but eliminated VSP repair activity in the cells. The remaining possible phenotype, M- Vsr+ was also constructed. When ClaI or AvaI fragments from the chromosomal DNA insert were deleted, the resulting plasmids were methylation defective but repair proficient (Fig. 1). It is clear from these data that the biochemical functions responsible for the two phenotypes are coded by adjacent segments of DNA. In other words, they are likely to be coded by separate
genes.
Sequence analysis of the dem locus. The E. coli chromosomal DNA present in pDCM23 was chosen for sequence analysis. Chromosomal DNA from pDCM23 and other Bal3l deletion derivatives was cloned into M13 phage vectors (39), and their sequence was determined by the chain termination technique (34). The strategy used for sequencing is summarized in Fig. 2A. Ninety-five percent of the sequence, including all of the open reading frames (ORFs) discussed below, was determined on both of the strands. The remain-
ing part of the sequence was derived from two overlapping sequencing runs for one of the strands (Fig. 2A). The sequence of the 2,472-bp insert in pDCM23 is presented in Fig. 3 (GenBank accession no. M32307). The sequence was analyzed for the presence of ORFs by use of the program DNA Strider (27) (Fig. 2B). Positions of four putative genes, i.e., ORFs starting with an AUG codon that are greater than 100 codons in length, are shown in Fig. 2C. The amino acid sequence of the ORFs labeled dcm and vsr are displayed in Fig. 3. The largest ORF that starts with an AUG codes for a 473-amino-acid protein with the predicted molecular weight of 53,431. There are two reasons to believe that this ORF is the dcm gene. The complementation analysis presented in Fig. 1 was consistent with this assignment. Among the Bal3M deletions from the right end of the insert, plasmids with larger inserts than pDCM8 showed full methylase activity, while those smaller than pDCM8 showed no methylase activity (see also Fig. 2). pDCM8 showed a level of methylase activity that was sufficient to protect some, but not all, of the Dcm sites within it. The DNA sequence analysis revealed that the C-terminal eight amino acids from the putative dcm gene had been deleted in the construction of pDCM8 (Fig. 3). The next larger derivative, pDCM21, contained the complete putative dem and was M+ (Fig. 3).
VOL. 172, 1990
GENE REQUIRED FOR VSP REPAIR IN E. COLI
4217
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Vol. 172, No. 8
A Gene Required for Very Short Patch Repair in Escherichia coli Is Adjacent to the DNA Cytosine Methylase Gene ANJUM SOHAIL,"2 MARGARET LIEB,3 MUBASHER DAR,4
AND
ASHOK S. BHAGWATl.4*
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 117241; Centre for Advanced Molecular Biology, University of the Punjab, Lahore-20, Pakistan2; University of Southern California School of Medicine, Los Angeles, California 900333; and Department of Chemistry, Wayne State University, Detroit, Michigan 482024 Received 14 March 1990/Accepted 9 May 1990
Deamination of 5-methylcytosine in DNA results in T/G mismatches. If unrepaired, these mistches can lead to C-to-T transition mutations. The very short patch (VSP) repair process in Escherichia coli counteracts the mutagenic process by repairing the mismatches in favor of the G-containing strand. Previosly we have shown that a plasmid containing an l1-kilobase fragment from the E. coli chromosome can complement a chromosomal mutation defective in both cytosine methylation and VSP repair. We have now mapped the regions essential for the two phenotypes. In the process, we have constructed plasmids that complement the chromosomal mutation for methylation, but not for repair, and vice versa. The genes responsible for these phenotypes have been identified by DNA sequence analysis. The gene essential for cytosine methylation, dem, is predicted to code for a 473-amino-acid protein and is not required for VSP repair. It is similar to other DNA cytosine methylases and shares extensive sequence similarity with its isoschizomer, EcoRl methylase. The segment of DNA essential for VSP repair contains a gene that should code for a 156-amino-acid protein. This gene, named vsr, is not essential for DNA methylation. Remarkably, the 5' end of this gene appears to overlap the 3' end of dcm. The two genes appear to be transcribed from a common promoter but are in different translational registers. This gene arrangement may assure that Vsr is produced along with Dcm and may minimize the mutagenic effects of cytosine methylation. Base mismatches can arise in DNA for a variety of These include replication errors and the action of chemical mutagens that react with DNA (for a review, see reference 30). An additional cause for the generation of base mismatches is the inherent instability of cytosine. Under physiological conditions, cytosine deaminates to uracil at appreciable rates (24). The resulting uracil/guanine mismatches are repaired by a process which, in the first step, involves the removal of uracil (23). 5-Methylcytosine is more unstable than cytosine (10, 24), and its deamination results in the creation of thymine/guanine (T/G) mismatches. If not correctly repaired prior to replication, C-to-T transitions occur. Cytosine methylation sites in DNA have been found to be "hotspots" for such mutations in Escherichia coli (7; M. Lieb, unpublished results). The only DNA cytosine methylase in E. coli K-12, Dcm, transfers a methyl group to position 5 of the internal cytosine in the sequence 5'-CCWGG-3', where W is A or T (29). E. coli also contains a base mismatch correction process that corrects T/G mismatches in sequence contexts such as Cl AGG/GjTCC and CITGG/GGACC to C/G (21). This process, called very short patch (VSP) repair, reduces the mutagenic effect of cytosine methylation (M. Lieb, unpublished results). It is likely that this process arose in E. coli to reduce the mutagenicity of 5-methylcytosine. Marinus and Morris used a chemical mutagen to obtain a series of E. coli mutations, dcm-1 through dcm-11, that were defective in the methylation of cytosines in DNA (28). One of these mutations, dcm-6, was later shown to lead to the loss of VSP repair in the cell as well (14, 20, 40). This led to the conclusion that Dcm was required for VSP repair and might be a bifunctional protein. We recently showed that a plasmid containing an 11-kilobase fragment of chromosomal
DNA from wild-type E. coli complements both the methylation and repair defects of dcm-6 (22). This is consistent with the suggestion that Dcm plays a direct role in VSP repair. In that study, we also tested the gene for another methylase, EcoRII, for its ability to complement dcm-6 for its repair defect. EcoRII methylase is part of the EcoRII restrictionmodification system and has sequence specificity identical to that of Dcm (29, 35). Hence, the gene that codes for the EcoRII methylase complements dcm-6 for methylation. Interestingly, it did not restore VSP repair in the cell (22). As a result of these observations, it was concluded that the ability of EcoRII to methylate Dcm/EcoRII sites is not sufficient for that enzyme to have a role in VSP repair. We report here that this is true of Dcm as well. Contrary to previous reports, dcm plays no direct role in VSP repair. Instead, a gene adjacent to it is required for this process. In turn, this gene appears to play no role in DNA methylation. (A preliminary report of this work was published [4] as the summary of a paper presented at the New England BioLabs Workshop on Biological DNA Modifications, Gloucester, Mass., 20 to 23 May 1988.)
reasons.
*
MATERIALS AND METHODS
Bacterial strains. GM1 [F- thr-l ara-14 leuB6 A(gptproA)62 lac Yl tsx-33 supE44 galK2 hisG4 rflhbD mgl-S1 rpsL31 kdgKSJ xyl-S mtl-l metBI thi-1]; its derivatives carrying various dcm alleles; GM30 (thr-J hisG4 leuB6 rpsL ara-14 supE44 lacYl tonA31 tsx-78 galK2 galE2 xylS thi-l mtl-i) and GM31 (i.e., GM30 dcm-6) were kindly provided by M. G. Marinus (University of Massachusetts School of Medicine, Amherst, Mass.). RP4182 [A(supD-dcm-fla) trp gal rpsL] was provided by J. S. Parkinson (University of Utah, Salt Lake City, Utah). Assay for methylation of Dcm sites. The ability of a bacterial strain to methylate Dcm/EcoRII sites was assessed
Corresponding author. 4214
GENE REQUIRED FOR VSP REPAIR IN E. COLI
VOL. 172, 1990
in two different ways. Plasmid DNAs from the strains
TABLE 1. VSP repair phenotypes of dcm mutations
were
routinely digested with EcoRII endonuclease (Bethesda Research Laboratory, Rockville, Md.), and the result was analyzed by electrophoresis on an agarose gel. Methylation of the Dcm/EcoRII sites results in the protection of the plasmid DNA against cleavage. Quantitation of the methylase activity was done for selected strains by determining the number of 3H atoms transferred from [3H]adenosyl-L-methionine (Dupont, NEN Research Products, Boston, Mass.) to chromosomal DNA from RP4182 as described before (5). Assay for VSP repair. The percentage of cI+ progeny among N+ O+ recombinants resulting from a cross of bacteriophage mutants Nam53 cIam6 O+ and N+ cI CP7 Oam29 was used to quantitate VSP repair as described before (21). Mutation am6 changes a CCAGG sequence to CTAGG; mutation CP7 is a T-to-A transversion located 20 base pairs (bp) to the left of am6 on the X map. Briefly, bacteria were infected with a 1:1 mixture of the two mutants to give a total multiplicity of infection of 10. After a 15-min adsorption period at 37°C, broth was added and incubation
continued for a total of 100 min, followed by the addition of CHCl3 and vortexing. Recombinants that are N+ O+ were selected by plating the phage progeny on a lawn of Supo bacteria; N+ O+ phage that are also cI+ form turbid plaques, while phage that carry the am6 or the CP7 mutation in cI form clear plaques. N+ O+ recombinants that are also cI+ require three crossover events and are expected to be very rare. However, during the recombinational events leading to N+ O+ phage, heteroduplex regions span the intervening cI gene, resulting in am6/+ (T/G) mismatches. Correction of am6 (T) to (C) results in excess N+ cI+ O recombinants. Therefore, the fraction of cI+ among the recombinant progeny phage is a measure of VSP repair in the host. Recombinant DNA work and DNA sequencing. Standard techniques were used in the construction of Bal3l-mediated deletions, deletion of restriction fragments, and construction of M13 phage clones (26). Inserts in the M13 clones were sequenced by using appropriate primers by the technique of Sanger et al. (34). was
RESULTS VSP repair and Dcm phenotypes can be unlinked. Three research groups have independently reported that E. coli K-12 cells carrying the dcm-6 mutation are deficient in VSP repair (14, 20, 40). dcm-6 is 1 of the 11 independent alleles of dcm isolated by Marinus and Morris by chemical mutagenesis of the strain GM1 (28). The nature of the mutation(s) is not known for any of the alleles. Three of the dcm mutant alleles have been lost; we have tested five of the remaining alleles for their effect on VSP repair. The results are summarized in Table 1. When a bacteriophage X cross diagnostic for VSP repair was done in GM1, the percentage of cI+ recombinants obtained was about 1.8% (Table 1). This number is similar to that obtained in another dcm' strain, GM30 (Table 1) (20). In GM31, the dcm-6 derivative of GM30, this number was sixfold lower (Table 1) (20), indicating a lower level of VSP repair. GM1 derivatives containing the alleles dcm-9 or dcm-10 showed a similarly low level of repair activity (Table 1). These mutations caused 10- and 12-fold reductions, respectively, in the Dcm activity in the cell (28). In contrast, GM1 derivatives carrying the alleles dcm4 or dcm-7 showed the same level of repair activity as GM1 and hence are VSP repair proficient (Table 1). Surprisingly, the derivative with the allele dcm-J showed slightly higher VSP repair activity
4215
Strain
GM30 GM31 GM1 GM5 GM8 GM11 GM13 GM14 RP4182
Allele
dcm' dcm-6
dcm' dcm-1 dcm4 dcm-7 dcm-9 dcm-1O Adcm
% of cI+ recombinantsa
2.2 0.36 1.8 6.0 2.0 2.5 0.32 0.49 0.08
± ± ± ±
+
± ± ± ±
0.6 0.15 0.3 1.9 0.5 0.4 0.19 0.07 0.03
a Among N+ O+ progeny (see Materials and Methods). Each value is an average of data from three or more experiments.
than GM1. Mutations dcm-J, dcm4, and dcm-7 cause 6-, 500-, and 2-fold reductions, respectively, in the Dcm activity in the cell (28). It is clear that there is no direct correlation between the Dcm activity and VSP repair activity in these strains. From these data we concluded that, although Dcm and VSP repair are tightly linked to each other, the two phenotypes can be unlinked. It should also be noted that there may not be a complete loss of VSP repair activity in dcm-6-, dcm-9-, or dcm10-containing strains. RP4182 is a strain of E. coli K-12 that carries a large deletion at about 43' on the chromosome and is missing several genes including dcm (2, 5, 17). It consistently produced three- to fourfold fewer cI+ recombinants in a similar cross (Table 1). It is possible that this is a result of the differences in the genetic backgrounds of GM1 and RP4182. It is more likely that there is some residual VSP repair in the strains containing the dcm-6, dcm-9, or dcm-10 alleles, and the reason for the lower numbers of cI+ recombinants in RP4182 is that the deletion in its chromosome eliminates a gene(s) essential for VSP repair. Dcm and VSP repair phenotypes can be separated biochemically. We have shown that a multicopy plasmid, pDCM1, that contains an 11-kilobase-pair DNA fragment from the E. coli chromosome (5), complements the dcm-6 mutation for VSP repair as well as methylation at the Dcm sites (22). Another plasmid, pDCM3, containing an overlapping chromosomal fragment (5), also complements this mutation for methylation and repair (data not shown). pDCM4 was created by deleting a HindlIl fragment within pDCM3 (5). This plasmid also complements the dcm-6 mutation for both the phenotypes, i.e., it has the phenotype methylation-positive VSP repair-positive (M+ Vsr+) (22). Bal3l deletions were generated from three unique sites in pDCM4 (BamHI, StuI, and Sall), and the resulting derivatives were tested for methylation and VSP repair (Fig. 1). Short deletions that removed sequences from the right side of the chromosomal insert had little effect on either phenotype (pDCM22, pDCM23, and pDCM33). Larger leftward deletions from the BamHI site showed two new phenotypes, M+ Vsr- and M- Vsr- (Fig. 1). All constructs that had more than about 2.0 kilobases of DNA removed from the right end were Vsr- (i.e., pDCM8, pDCM9, and pDCM21). One of these plasmids, pDCM21, fully complemented the dcm-6 mutation for methylation. Another, pDCM8, showed a reduced level of methylation activity. DNA prepared from RP4182 containing this plasmid was only partially resistant to the EcoRII endonuclease (data not shown). Deletions from the right end that removed greater than 2.7 kilobase pairs of the insert were defective in methylation as well as in VSP repair (pDCM9; data not shown). The Vsr- phenotype could also be created by the insertion
4216
SOHAIL ET AL.
J. BACTERIOL.
Sail Sau3A/BamHI / Cial Plasmi
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4 4.3Okbp FIG. 1. Phenotypes of pDCM4 derivatives. Various derivatives of pDCM4 (5) were constructed by Bal3l-mediated deletions, by the deletion of restriction fragments, or by the insertion of a linker. Only those derivatives that were tested for their methylation (M) and VSP repair (VSR) abilities are shown here. Others were used for the sequencing of the DNA segment (see below). Most of the pBR322-derived DNA in these plasmids is not shown. Filled-in rectangles represent DNA derived from the E. coli chromosome; the rest is from pBR322. Plasmids pDCM8, pDCM22, and pDCM23 were generated by Bal3l-mediated deletions from the BamHI site. PDCM9 was generated by ligating a BamHI linker at the StuI site and deleting the smaller BamHI fragment from the plasmid. After the deletion of DNA, BamHI linkers were ligated to the ends and the plasmids were circularized. Plasmid pDCM24 was made by Bal31-mediated deletion from the SalI site. No linker was inserted at the end prior to circularization, and the precise endpoint of deletion is unknown. Hence, the endpoint is indicated by a broken line. Plasmid pDCM33 was made by cloning a SphI-BamHI fragment from pDCM22 into pACYC184. The SphI site used lies within the pBR322-derived DNA in pDCM33. Deletion of AvaI and ClaI fragments from pDCM23 created pDCM28 and pDCM36, respectively. The construction of pDCM35 is described in the text. The ability (or inability) of these plasmids to complement dcm-6 for VSP repair and for methylation of Dcm/EcoRII sites is indicated by a plus or minus sign under the columns labeled VSR and M, respectively. The protection of Dcm/EcoRII sites, as determined by the gel electrophoretic assay, was the criterion used to judge the methylation phenotype. A frequency of cI+ recombinants below 0.5% in standard phage crosses was judged to be indicative of no VSP repair. Cells carrying plasmids indicated to be Vsr+ generally showed a frequency of cI+ recombinants in excess of 4%. 1
of 4 bp at the unique NcoI site in the chromosomal insert (Fig. 1, pDCM35). This was achieved by cutting the plasmid pDCM23 with NcoI and filling in the four nucleotide cohesive tails with DNA polymerase. Success of the fill-in reaction was tested by checking the self-ligated molecules for a new NslI site. This frameshift mutation did not affect the methylation ability of the plasmid but eliminated VSP repair activity in the cells. The remaining possible phenotype, M- Vsr+ was also constructed. When ClaI or AvaI fragments from the chromosomal DNA insert were deleted, the resulting plasmids were methylation defective but repair proficient (Fig. 1). It is clear from these data that the biochemical functions responsible for the two phenotypes are coded by adjacent segments of DNA. In other words, they are likely to be coded by separate
genes.
Sequence analysis of the dem locus. The E. coli chromosomal DNA present in pDCM23 was chosen for sequence analysis. Chromosomal DNA from pDCM23 and other Bal3l deletion derivatives was cloned into M13 phage vectors (39), and their sequence was determined by the chain termination technique (34). The strategy used for sequencing is summarized in Fig. 2A. Ninety-five percent of the sequence, including all of the open reading frames (ORFs) discussed below, was determined on both of the strands. The remain-
ing part of the sequence was derived from two overlapping sequencing runs for one of the strands (Fig. 2A). The sequence of the 2,472-bp insert in pDCM23 is presented in Fig. 3 (GenBank accession no. M32307). The sequence was analyzed for the presence of ORFs by use of the program DNA Strider (27) (Fig. 2B). Positions of four putative genes, i.e., ORFs starting with an AUG codon that are greater than 100 codons in length, are shown in Fig. 2C. The amino acid sequence of the ORFs labeled dcm and vsr are displayed in Fig. 3. The largest ORF that starts with an AUG codes for a 473-amino-acid protein with the predicted molecular weight of 53,431. There are two reasons to believe that this ORF is the dcm gene. The complementation analysis presented in Fig. 1 was consistent with this assignment. Among the Bal3M deletions from the right end of the insert, plasmids with larger inserts than pDCM8 showed full methylase activity, while those smaller than pDCM8 showed no methylase activity (see also Fig. 2). pDCM8 showed a level of methylase activity that was sufficient to protect some, but not all, of the Dcm sites within it. The DNA sequence analysis revealed that the C-terminal eight amino acids from the putative dcm gene had been deleted in the construction of pDCM8 (Fig. 3). The next larger derivative, pDCM21, contained the complete putative dem and was M+ (Fig. 3).
VOL. 172, 1990
GENE REQUIRED FOR VSP REPAIR IN E. COLI
4217
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