Mutations in the Bacillus subtilis beta clamp that separate its roles in DNA replication from mismatch repair

Abstract

The beta clamp is an essential replication sliding clamp required for processive DNA synthesis. The beta clamp is also critical for several additional aspects of DNA metabolism, including DNA mismatch repair (MMR). The dnaN5 allele of Bacillus subtilis encodes a mutant form of beta clamp containing the G73R substitution. Cells with the dnaN5 allele are temperature sensitive for growth due to a defect in DNA replication at 49 degrees C, and they show an increase in mutation frequency caused by a partial defect in MMR at permissive temperatures. We selected for intragenic suppressors of dnaN5 that rescued viability at 49 degrees C to determine if the DNA replication defect could be separated from the MMR defect. We isolated three intragenic suppressors of dnaN5 that restored growth at the nonpermissive temperature while maintaining an increase in mutation frequency. All three dnaN alleles encoded the G73R substitution along with one of three novel missense mutations. The missense mutations isolated were S22P, S181G, and E346K. Of these, S181G and E346K are located near the hydrophobic cleft of the beta clamp, a common site occupied by proteins that bind the beta clamp. Using several methods, we show that the increase in mutation frequency resulting from each dnaN allele is linked to a defect in MMR. Moreover, we found that S181G and E346K allowed growth at elevated temperatures and did not have an appreciable effect on mutation frequency when separated from G73R. Thus, we found that specific residue changes in the B. subtilis beta clamp separate the role of the beta clamp in DNA replication from its role in MMR.

FIG. 1.

The β clamp^G73R protein accumulates in vivo. (A) Growth of wild-type PY79 and isogenic strains with the dnaN5, dnaN34, and dnaB134 alleles. Tenfold serial dilutions of each strain were plated and grown at the indicated temperatures. Pictures were captured following 14 h of growth. Representative plates from several experiments are shown. (B) Relative mutation frequencies (n-fold increases above the wild-type level) of strains carrying the dnaB134, dnaN5(G73R), and dnaN34 alleles determined at 37°C. The histogram represents the mean ± the standard error of the mean from at least four independent experiments. (C) Representative immunoblot assay of the β clamp^G73R mutant and wild-type β clamp proteins at 30°C and following a temperature shift to 49°C for the times indicated. The total loaded sample was normalized to cell number based on the optical density of each culture. (D) Ribbon model of the B. subtilis β clamp. One protomer is cyan, and the second protomer is green. The predicated location of the G73R mutation is shown as red spheres.

FIG. 2.

Disruption of Y family DNA polymerases does not alter the mutagenesis caused by dnaN5(G73R). The mean relative mutation frequencies of strains with the indicated alleles are shown in the histogram. Error bars show the standard error of the mean for 8 to 17 independent experiments. The mutation frequency of each strain was normalized to the mutation frequency of the wild-type control strain PY79.

FIG. 3.

dnaN5(G73R) causes slight overinitiation of DNA replication. For all of the images, Spo0J-GFP is green and FM4-64 vital membrane staining is red. Cells were imaged during exponential growth (optical density at 600 nm of ∼0.5) according to Materials and Methods. Panels: A, wild-type dnaN⁺; B, dnaN5(G73R) at 30°C; C, dnaN5(G73R) at 49°C; D, yabA::cat; E, scoring of the percentage of cells with n Spo0J-GFP foci in the indicated genetic backgrounds and temperatures as shown. The number of cells scored is also indicated. Bar, 3 μm.

FIG. 4.

Intragenic suppressors restore growth of B. subtilis at 49°C. Shown is the growth of the wild-type and isogenic strains with the indicated alleles [dnaN(G73R) and suppressors] at 30°C and 49°C following 10-fold serial dilution and plating on LB agar plates. Pictures were taken following 14 h of growth. Representative plates from several experiments are shown.

FIG. 5.

Locations of intragenic suppressors of dnaN5(G73R). (A) Ribbon diagram of the B. subtilis β clamp homology model. One protomer is cyan, and the other is green. The predicted positions of S22P, G73R, S181G, and E346K are shown in red, and the amino acids comprising the hydrophobic cleft are white. (B) Representative immunoblot assay of each β clamp protein indicated from cultures grown at 30°C. The loaded sample was normalized to cell number. (C) Comparison of the amino acid substitutions examined in this study and the corresponding E. coli β clamp residue based on alignment of the primary structures. The mutation-bearing allele, if it is known, is in parentheses.

FIG. 6.

Intragenic suppressors of dnaN5(G73R) maintain an MMR-dependent increase in mutation frequency. (A) The mean relative mutation frequencies of strains with the indicated alleles are presented. The mutation frequency of each strain was normalized to the mutation frequency of wild-type control strain PY79. (B) Relative mutation frequency of each strain indicated after the introduction of an MMR-defective allele (mutSL::spc) in combination with each dnaN allele. For comparison, we show these relative to the mild dominant negative effect of mutL with the A17T missense mutation expressed from the amyE locus under P_spac control. (C) Relative mutation frequencies of the dnaN5(G73R) mutant, the mutS5A mutant, and the double mutant. The bars in the histograms represent the mean ± the standard error of the mean for 6 to 20 independent experiments.

FIG. 7.

Missense mutations S181G and E346K restore growth of B. subtilis at 49°C. Shown is the growth of each strain with the dnaN allele indicated under P_spac control at the native locus following 10-fold serial dilutions and at the temperatures indicated. Representative plates from several experiments are shown.

FIG. 8.

dnaN alleles are restored for support of MutS-GFP focus formation. Panels A, C, and E are differential interference contrast images showing cell boundaries. Images B, D, and F are MutS-GFP foci following treatment with 2-AP at 600 μg/ml for 1 h. Panels: A and B, MutS-GFP, dnaN⁺, 2-AP; C and D, MutS-GFP, dnaN5(G73R), 2-AP; E and F, MutS-GFP, dnaN(G73R, E346K), 2-AP. Bar, 3 μm.