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. Author manuscript; available in PMC: 2015 Mar 1.
Published in final edited form as: Mutat Res. 2014 Feb 1;761:21–33. doi: 10.1016/j.mrfmmm.2014.01.005

Fig. 2.

Fig. 2

Effect of mutations in the chromosomal polA gene on spontaneous mutagenesis in recA730 lexA(Def) ΔumuDCΔ dinBΔ mutL strains harboring the empty vector, pGB2, or pRW134 expressing wild-type pol V, or JM963 expressing UmuD′ and UmuC_Y11A. A. The location of the amino acid substitutions of each respective missense polA allele are indicated within the structural domains of pol I. The polA_ΔC allele expresses a truncated pol I protein (residues 1–768). B. Western Blot of steady-state levels of pol I in various strains of E. coli. Whole-cell extracts were obtained from the following strains: RW710 (polA+); RW900 (ΔpolA::Kan); (RW1088 (polA107); RW1048 (polA_D424A); RW1042 (polA_F769A/F771A); RW1098 (polA_ΔC) and the steady-state levels of pol I determined as described in Materials and methods. As observed, the steady state levels of pol I encoded by the three missense polA alleles were similar to the level of wild-type pol I. However the steady-state level of the truncated pol I (1–768) protein (encoded by polA_ΔC) was significantly lower compared to the wild-type enzyme and missense pol I mutants and was barely detectable unless the images were greatly overexposed (not shown). C. Spontaneous mutagenesis was measured by assaying reversion of the hisG4 ochre allele (leading to histidine prototroph) as described in Materials and methods. The following mismatch repair-defective strains were used in the assays: RW710 (polA+); RW1088 (polA107); RW1050 (Δxni); RW1054 (polA107/Δxni); RW1048 (polA_D424A); RW1042 (polA_F769A/F771A); RW1098 (polA_ΔC). The average number of His+ revertants per plate ± standard error of the mean (SEM) for the strains lacking pol V, or expressing wild type, or umuC_ Y11A pol V is indicated in the table below the graph. Since the extent of mutagenesis promoted by wild-type pol V differs in the various polA strains, the level of mutagenesis promoted by umuC_ Y11A is expressed as a percentage of wild-type pol V-dependent mutagenesis in the same strain and shown in the graph. Comparison of these values allows us to characterize the effect of mutations in pol I on the excision repair of ribonucleotides incorporated by the umuC_ Y11A allele of pol V. The difference in umuC_ Y11A-dependent mutagenesis between polA+ and either polA 3′→5′ exo− or polA_ΔC strains are statistically significant (p values of 0.02 and 0.015, respectively) as revealed by Student’s t-test analysis.