Investigating the mechanisms of ribonucleotide excision repair in Escherichia coli

Abstract

Low fidelity Escherichia coli DNA polymerase V (pol V/UmuD'2C) is best characterized for its ability to perform translesion synthesis (TLS). However, in recA730 lexA(Def) strains, the enzyme is expressed under optimal conditions allowing it to compete with the cell's replicase for access to undamaged chromosomal DNA and leads to a substantial increase in spontaneous mutagenesis. We have recently shown that a Y11A substitution in the "steric gate" residue of UmuC reduces both base and sugar selectivity of pol V, but instead of generating an increased number of spontaneous mutations, strains expressing umuC_Y11A are poorly mutable in vivo. This phenotype is attributed to efficient RNase HII-initiated repair of the misincorporated ribonucleotides that concomitantly removes adjacent misincorporated deoxyribonucleotides. We have utilized the ability of the pol V steric gate mutant to promote incorporation of large numbers of errant ribonucleotides into the E. coli genome to investigate the fundamental mechanisms underlying ribonucleotide excision repair (RER). Here, we demonstrate that RER is normally facilitated by DNA polymerase I (pol I) via classical "nick translation". In vitro, pol I displaces 1-3 nucleotides of the RNA/DNA hybrid and through its 5'→3' (exo/endo) nuclease activity releases ribo- and deoxyribonucleotides from DNA. In vivo, umuC_Y11A-dependent mutagenesis changes significantly in polymerase-deficient, or proofreading-deficient polA strains, indicating a pivotal role for pol I in ribonucleotide excision repair (RER). However, there is also considerable redundancy in the RER pathway in E. coli. Pol I's strand displacement and FLAP-exo/endonuclease activities can be facilitated by alternate enzymes, while the DNA polymerization step can be assumed by high-fidelity pol III. We conclude that RNase HII and pol I normally act to minimize the genomic instability that is generated through errant ribonucleotide incorporation, but that the "nick-translation" activities encoded by the single pol I polypeptide can be undertaken by a variety of back-up enzymes.

Keywords: RNase H; Ribonucleotide excision repair; Steric gate mutant; UmuC: spontaneous mutagenesis; Y-family DNA polymerase.

Fig. 1

Enzymatic activities of pol I on the FLAP endo-, or exonuclease DNA substrates containing a 5′-terminal monoribonucleotide on the downstream blocking oligonucleotide. The pol I-catalyzed strand displacement synthesis and 3′→5′ exonucleolytic proofreading were assayed using nicked DNA substrate generated by annealing of the 49-mer DNA template containing an abasic site (X) with the P³²-labeled 17-mer primer and the 32-mer downstream blocker (A). The nick-specific exonuclease (B) and FLAP-specific endonuclease (C) activities of pol I were visualized using the 17- or 27-mer primers, respectively, hybridized together with the P³²-labeled 32-mer blocker to the 49-mer DNA template. Structures of DNA substrates are schematically presented on the top of each gel image (brown – template, red – primer, blue - blocker). In substrate I, the 17mer primer is labeled, while in substrates II and III the 32mer blocking oligonucleotide is labeled (as indicated by, *). The arrows point to the cleavage sites on the DNA made by 3′→5′ exo- (purple ⇪ in I), 5′→3′exo- (green ▼ in II), and flap endonuclease (orange ▼ in II & III) activities of pol I and the corresponding exonucleolytic products, are indicated on the gels. The 27-mer bands seen on the gel in the panel A correspond to the products of primer extension blocked by the abasic site, while the 49-mer band correspond to the primer extensions past the lesion to the full-size product (TLS – translesion synthesis). Reactions were performed using DNA substrate (2 nM), purified pol I (4, 16, 63, 250 pM, or 1 nM), and mixtures of all four dNTPs (100 μM) for 15 min and analyzed by polyacrylamide gel electrophoresis as described in the Materials and Methods section.

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.

Fig. 3

Effect of Rnase HII on the level of spontaneous mutagenesis promoted by the empty vector, pGB2, or pRW134 expressing wild-type pol V, or JM963 expressing UmuD′ and UmuC_Y11A in various polArecA730 lexA (Def) ΔumuDCΔ dinBΔ mutL ± ΔrnhB strains. Spontaneous mutagenesis was measured by assaying reversion of the hisG4 ochre allele (leading to histidine prototrophy) as described in Experimental procedures. 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. The average 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 are taken from [14] The following mismatch repair-defective strains were used in the assays: RW710 (polA⁺); RW1056 (polA⁺Δ rnhB); RW1048 (polA_D424A); RW1320 (polA_D424A ΔrnhB); RW1098 (polA_ΔC); RW1336 (polA_ΔC ΔrnhB). The data for the polA⁺, polA 3′→5′ exo⁻ and polA_ΔC strains expressing wild-type RNase HII are taken from Fig. 2 and are shown for direct comparison with the data for the ΔrnhB strains. The difference in umuC_ Y11A-dependent mutagenesis is statistically significant for the polA⁺ and polA⁺/ΔrnhB (p=0.02) and for polA_ΔC and polA_ΔC/ΔrnhB (p<0.002) strains.

Fig 4

Spectra of spontaneously arising rpoB mutations in recA730 lexA(Def) ΔdinB ΔumuDC ΔmutLrnhB +/− strains expressing umuC_Y11A. A. polA _D424A strains deficient in pol I 3′→5′ exonuclease activity. The arrows indicate mutagenic hot spots within the rpoB. B. polA _ΔC strains deficient in pol Ipolymerase activity. Approximately 300 Rif ^R mutants were analyzed for each strain (Table 2). The forward mutation rates were assayed by measuring resistance to rifampicin and were in the range of 3–5 × 10⁻⁶. The spectra in the rnhB+ and ΔrnhB strains differ in both the types of mutations and locations of mutagenic hot-spots. Because of defects in MMR, all the spectra are dominated by transition mutations. However, since low-fidelity pol V makes a significant number of transversion mutations compared to other E. coli DNA polymerases [34, 37, 52], the number of umuC_Y11A-dependent transversions in the rnhB strains is higher than in the rnhB⁺ strains (see Table 2). The shift in the types of mutations observed is significantly more pronounced in the polA_ΔC strains. The rnhB⁺ strains undergo active RER; as a consequence, they have a reduced number of pol V-specific transversions, and the spectrum generated in these strains reflect uncorrected errors made by pol I (panel A) or pol III (panel B) during the DNA re-synthesis step of RER.

Fig. 5

Effect of polBex1 and mutD5 alleles on spontaneous mutagenesis in recA730 lexA(Def) ΔumuDCΔ dinBΔ mutLpolA ⁺/ polA_ΔC strains harboring the empty vector, pGB2, or pRW134 expressing wild-type pol V, or JM963 expressing UmuD′ and UmuC_Y11A. A. Full-length polA encodes a 928 amino acid protein, whereas the polA_ΔC allele expresses a truncated pol I protein (residues 1–768) that lacks key structural domains necessary to catalyze DNA synthesis. B. Because of their low viability on the defined “low histidine” minimal plates, spontaneous mutagenesis was measured by assaying reversion of the hisG4 ochre allele (leading to histidine prototrophy) on minimal agar plates containing 4μg/ml Casamino acids as described in Materials and methods. The following mismatch repair-defective strains were used in the assays: RW1236 (polA⁺); RW1098 (polA_ΔC); RW1226 (polA⁺polBex1 ); RW1324 (polA_ΔC polBex1); RW1250 (polA⁺mutD5 ); RW1218 (polA_ΔC mutD5). 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 isogenic 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 inactivating the proofreading activity of pol II (polBex1), or proofreading-deficient pol III (mutD5), on the excision repair of ribonucleotides incorporated by the umuC_ Y11A allele of pol V in polA⁺ or polA_ΔC strains.

Fig. 6

A model for ribonucleotide excision repair in E. coli. Pol V introduces multiple base substitutions (shown as d in orange) when it gains access to undamaged chromosomal DNA. It can also incorporate ribonucleotides (shown as r in red) into the nascent DNA strand. RNase HII incises the bond at the rNMP/dNMP junction 5′ to the ribonucleotide, generating DNA containing a single stranded break with 5′-phospho-ribonucleotide and 3′-hydroxyl ends. Pol I commences DNA synthesis at the nick and promotes strand displacement of the mutagenic pol V tract (shown as d in green). In polA mutants that are unable to promote strand displacement, the unwinding of the nicked intermediate proceeds through the action of an unknown DNA helicase, or polymerase (shown in parentheses). In polA_ΔC strains deficient for DNA synthesis, the 3′-hydroxyl end of the nicked intermediate is extended by high-fidelity pol III (shown in parentheses). The displaced region containing rNMP and misincorporated dNMPs is usually degraded by the 5′→3′ exonuclease or 5′-FLAP endonuclease activity of pol I, but in its absence, the displaced strand may be degraded by the FLAP endonuclease Xni, or another (yet to be identified) single-stranded nuclease (shown in parentheses). Complete repair occurs once the nick is sealed by DNA ligase. Although this model is based upon our present studies with the error-prone umuC_Y11A steric gate mutant of pol V, we envisage that identical processes occur when high-fidelity DNA polymerases inadvertently incorporate ribonucleotides into genomic DNA.