umuC Mutant Research Articles

RecFOR proteins are essential for Pol V-mediated translesion synthesis and mutagenesis

When the replication fork moves through the template DNA containing lesions, daughter-strand gaps are formed opposite lesion sites. These gaps are subsequently filled-in either by translesion synthesis (TLS) or by homologous recombination. RecA filaments formed within these gaps are key intermediates for both of the gap-filling pathways. For instance, Pol V, the major lesion bypass polymerase in Escherichia coli, requires a functional interaction with the tip of the RecA filament. Here, we show that all three recombination mediator proteins RecFOR are needed to build a functionally competent RecA filament that supports efficient Pol V-mediated TLS in the presence of ssDNA-binding protein (SSB). A positive contribution of RecF protein to Pol V lesion bypass is demonstrated. When Pol III and Pol V are both present, Pol III imparts a negative effect on Pol V-mediated lesion bypass that is counteracted by the combined action of RecFOR and SSB. Mutations in recF, recO or recR gene abolish induced mutagenesis in E. coli.

Altered translesion synthesis in E. coli Pol V mutants selected for increased recombination inhibition

Replication of damaged DNA, also termed as translesion synthesis (TLS), involves specialized DNA polymerases that bypass DNA lesions. In Escherichia coli, although TLS can involve one or a combination of DNA polymerases depending on the nature of the lesion, it generally requires the Pol V DNA polymerase (formed by two SOS proteins, UmuD′ and UmuC) and the RecA protein. In addition to being an essential component of translesion DNA synthesis, Pol V is also an antagonist of RecA-mediated recombination. We have recently isolated umuD′ and umuC mutants on the basis of their increased capacity to inhibit homologous recombination. Despite the capacity of these mutants to form a Pol V complex and to interact with the RecA polymer, most of them exhibit a defect in TLS. Here, we further characterize the TLS activity of these Pol V mutants in vivo by measuring the extent of error-free and mutagenic bypass at a single (6-4)TT lesion located in double stranded plasmid DNA. TLS is markedly decreased in most Pol V mutants that we analyzed (8/9) with the exception of one UmuC mutant (F287L) that exhibits wild-type bypass activity. Somewhat unexpectedly, Pol V mutants that are partially deficient in TLS are more severely affected in mutagenic bypass compared to error-free synthesis. The defect in bypass activity of the Pol V mutant polymerases is discussed in light of the location of the respective mutations in the 3D structure of UmuD′ and the DinB/UmuC homologous protein Dpo4 of Sulfolobus solfataricus.

Specific in vivo protein-protein interactions between Escherichia coli SOS mutagenesis proteins.

One of the components of the RecA-LexA-controlled SOS response in Escherichia coli cells is an inducible error-prone DNA replication pathway that results in a substantial increase in the mutation rate. It is believed that error-prone DNA synthesis is performed by a multiprotein complex that is formed by UmuC, UmuD', RecA, and probably DNA polymerase III holoenzyme. It is postulated that the formation of such a complex requires specific interactions between these proteins. We have analyzed the specific protein-protein interactions between UmuC, UmuD, and UmuD' fusion proteins, using a Saccharomyces cerevisiae two-hybrid system. In agreement with previous in vitro data, we have shown that UmuD and UmuD' are able to form both homodimers (UmuD-UmuD and UmuD'-UmuD') and a heterodimer (UmuD-UmuD'). Our data show that UmuC fusion protein is capable of interacting exclusively with UmuD' and not with UmuD. Thus, posttranslational processing of UmuD into UmuD' is a critical step in SOS mutagenesis, enabling only the latter protein to interact with UmuC. Our data seem to indicate that the integrity of the entire UmuC sequence is essential for UmuC-UmuD' heterotypic interaction. Finally, in our studies, we used three different UmuC mutant proteins: UmuC25, UmuC36, and UmuC104. We have found that UmuC25 and UmuC36 are not capable of associating with UmuD'. In contrast, UmuC104 protein interacts with UmuD' protein with an efficiency identical to that of the wild-type protein. We postulate that UmuC104 protein might be defective in interaction with another, unknown protein essential for the SOS mutagenesis pathway.

Role of ruvAB genes in UV- and γ-radiation and chemical mutagenesis in Escherichia coli

Escherichia coli umuC122:: Tn5 was mutagenized with N-methyl- N′-nitro- N-nitrosoguanidine to isolate mutations that block the residual γ-radiation mutagenesis observed in umuC strains. Two of these mutations were shown by transductional mapping and plasmid complementation to map in the ruvA and ruvB genes (i.e., ruvA200 and ruvB201). Whereas ruvA200 was complemented by ruvA + plasmids, the only other known ruvA mutation, ruvA59:: Tn 10 required both the ruvA + and ruvB + genes to show complementation. The ruvA200, ruvB201, ruvA59:: Tn 10 and ruvB60:: Tn 10 mutations all reduced γ-radiation-induced ochre reversion [ argE3(Oc) → Arg + ]] to about 30% of the wild-type level, and they all reduced UV-radiation-induced ochre reversion to about 15% of the wild-type level. The ruvA200 and ruvB201 mutants also showed reduced γ- and UV-radiation mutagenesis with two other assays [ hisG4(Oc) → His + and Rif s → Rif r]. Streptozotocin mutagenesis (Rif r was reduced to about half of the wild-type level in ruv strains, but ethyl methanesulfonate mutagenesis was normal. While the umuC strain did not show the oxygen enhancement of γ-radiation mutagenesis, the ruvA200 strain showed an oxygen effect that was similar to that shown by the wild-type strain. When the ruvA200 mutation was combined with the umuC mutation, γ-radiation mutagenesis was further reduced to 5% of the wild-type level and cells showed a synergistic sensitization to UV- and γ-radiation-induced killing. A mutational spectrum analysis indicates a general depression of both umuC-dependent and umuC-independent γ-radiation mutagenesis in the ruvA strain, which is in contrast with the site-specific reduction in γ-radiation mutagenesis that is observed in the umuC mutant. The reduced radiation mutagenesis in the ruvA strain could not be correlated with a reduction in transcription of the recA or umuC genes.

UmuC function is not essential for the production of all targeted lacI mutations induced by ultraviolet light.

Up to a quarter or more of the normal yield of lacI- mutations could be induced by ultraviolet light in a uvrA6 umuC122:: Tn5 strain if they were detected by plating on 5-bromo-4-chloro-3-indolyl-beta-D-galactoside medium, where all surviving cells can form colonies. Using phenyl beta-D-galactoside selection, which curtails post-irradiation growth, only low yields of mutations were induced. Nucleotide sequence analysis of 134 spontaneous and 145 ultraviolet light-induced mutations shows that broadly similar kinds of mutations were induced in the umuC mutant and its uvrA6 umuC+ counterpart. In particular, these data offer no reason for believing that most of the mutations induced in the umuC mutant were other than normal, targeted events. We conclude that UmuC function, rather than being essential, facilitates recovery and specifically, following the model of Bridges & Woodgate, that it facilitates the prompt resumption of chain elongation.

Post-replication repair and recombination in uvrA umuC strains of Escherichia coli are enhanced by vanillin, an antimutagenic compound

Effects of vanillin on UV killing of umuC mutant strains of E. coli were investigated in order to analyze the antimutagenic role of vanillin in mutagenesis. UV-irradiated uvrA umuC cells showed higher survival when plated on medium containing vanillin rather than medium without vanillin. This increased survival associated with exposure to vanillin was observed more clearly in uvrA umuC lexA(ind −) and uvrA umuC recF strains. However, the effect was inhibited by additional recB recC mutations and completely blocked by an additional recA mutation. As far as tested the increased survival of UV-treated cells by vanillin was dependent on a capacity for genetic recombination. The effect of vanillin on recombination frequency between 2 plasmid DNA, pATH4 (Cm r Tc s) and pBMX7 (Ap r Tc s), in a uvrA umuC background was investigated. A significantly higher frequency of plasmid recombination was observed when vanillin was present in the culture medium. These findings suggest that the antimutagenic effect of vanillin is the result of enhancement of a recA-dependent, error-free, pathway of post-replication repair.

Effect of DNA sequence changes on UV mutability of a purine anticodon triplet of glyU cloned on M13 phage.

Mutant forms of the glyU (glycyl tRNA) gene cloned in M13mp8 were subjected to uninduced targeted UV mutagenesis; i.e. phage particles were irradiated and used to infect unirradiated umuC+ or irradiated umuC mutant cells. The irradiated phage carried GAG at the anticodon triplet and transitions to GAA were scored. The uninduced targeted mutation rate was reduced by altering the sequence of the gene in the vicinity of the target purine (Pu) residue. In particular a triplet of pyrimidines (PyPyPy) 5' to the target G was changed to PyPuPy in order to prevent formation of cyclcobutane and 6-4 pyrimidine dimers close to the target. On this basis we suggest a mechanism for one type of uninduced regionally targeted UV mutagenesis.

Quantitation of the Involvement of the recA, recB, recC, recF, recJ, recN, lexA, radA, radB, uvrD, and umuC Genes in the Repair of X-Ray-Induced DNA Double-Strand Breaks in Escherichia coli

Isogenic Escherichia coli strains carrying single DNA-repair mutations were compared for their capacity for (i) the repair of X-ray-induced DNA double-strand breaks (DSB) as measured using neutral sucrose gradients; (ii) medium-dependent resistance, i.e., a recA-dependent X-ray survival phenomenon that correlates closely with the capacity for repairing DSB; and (iii) the growth medium-dependent, recA-dependent repair of X-ray-induced DNA single-strand breaks (SSB) as measured using alkaline sucrose gradients (about 80% of these SSB are actually parts of DSB). These three capacities were measured to quantitate more accurately the involvement of the various genes in the repair of DSB over a wide dose range. The mutations tested were grouped into five classes according to their effect on the repair of X-ray-induced DSB: (I) the recA, recB, recC, and lexA mutants were completely deficient; (II) the radB and recN mutants were about 90% deficient; (III) the recF and recJ mutants were about 70% deficient; (IV) the radA and uvrD mutants were about 30% deficient; and (V) the umuC mutant resembled the wild-type strains in its capacity for the repair of DSB.

Effect of an umuC mutation on phage λ induction

A possible role of the umuC gene product in the induction of the SOS responses was examined. We compared the expression of a genetic fusion, in which gene lacZ, encoding beta-galactosidase in Escherichia coli is under the direct control of the cI repressor from prophage lambda, in a umuC+ strain and in an otherwise isogenic umuC- mutant. We found that two times higher UV doses were required to obtain a similar induction in the umuC+ strain as in the umuC mutant. In addition we showed that, at the same UV dose after a lag period, the specific activity of beta-galactosidase increased more rapidly in the umuC mutant. We suggest that the wild-type umuC gene product participates in the processing of the SOS inducing structures caused by UV irradiation or prevents formation of some of them. This is compatible with a role of the UmuC protein in DNA synthesis past a replication block.

Genes from plasmid pKM101 in Haemophilus influenzae: separation of functions of mucA and mucB.

Haemophilus influenzae, normally not mutable by UV, became UV mutable with a recombinant plasmid insertion. A 7.8-kilobase-pair (kbp) fragment of the plasmid pKM101 containing the mucA and mucB genes was ligated to the shuttle vector pDM2, and a Rec- strain of H. influenzae was transformed with the ligated mixture. All of the transformants, unlike the parent Rec- strain, were resistant to UV, could carry out postreplication repair and Weigle reactivation, showed greatly increased spontaneous mutation, and contained a plasmid carrying an insert of only 1.2 rather than 7.8 kbp. This plasmid in a umuC mutant strain of Escherichia coli complemented a pKM101 derivative lacking mucA function but with an intact mucB gene, although there was no complementation with a mucA+ mucB- plasmid, suggesting that the newly constructed plasmid coded for the mucA protein; this is in accord with the restriction analysis and hybridization between the plasmid and a probe containing all of the mucA gene but only a small fraction of mucB. When one of the H. influenzae Rec- transformants lost the plasmid, the resistance to UV was retained but the high spontaneous mutation and UV mutability were not. The fact that there was hybridization between the chromosome of the "cured" strain and a probe containing both muc genes but none when almost no mucB was present suggested that at least part of the mucB gene had been integrated into the Rec- chromosome. Five different postreplication repair-proficient strains became UV mutable and had high spontaneous mutation rates caused by the putative mucA plasmid, indicating that these strains already possessed a chromosomal equivalent of the mucB gene.

Effect of the plasmid ColIb-P9 on cellular processes related to DNA repair.

The plasmid ColIb-P9 introduced into Escherichia coli K12 umuC mutant cells suppresses the deficiencies in mutagenesis and repair of mutants after UV-irradiation. These data suggest that ColIb-P9 encodes a product with a function similar to that of the chromosomal gene umuC. Tn5 insertion mutants of ColIb-P9 were isolated with an altered ability to restore UV-mutagenesis in the umuC mutant. The same plasmid mutations were shown to eliminate the effects of ColIb-P9 on UV-mutagenesis, survival after UV and mitomycin C treatment, reactivation of UV-irradiated lambda in unirradiated cells, Weigle-reactivation, induction of colicin E1 synthesis. The ColIb-P9 genes responsible for the enhancement of UV-mutagenesis were cloned within a 14 Md SalI fragment. Their location was established by restriction analysis of the mutant plasmid ColIb 6-13::Tn5. While the action of the plasmids ColIb-P9 and pKM101 is similar, these plasmids were shown to have opposite effects on cell survival and colicin E1 synthesis after mitomycin C treatment. A study of the mutant plasmids ColIb::Tn5 and pGW12 (muc- mutant of pKM101) has shown the difference in the effects of ColIb-P9 and pKM101 to be associated with the plasmid genes responsible for the protective and mutagenesis-enhancing effects of these plasmids in UV-irradiated cells.

Genetic activity of bleomycin in Escherichia coli

The induction of umuC gene expression, cell lethality, induction of W-reactivation of UV-irradiated λ-phage and the induction of mutagenesis caused by bleomycin (Blm) were studied in Escherichia coli K-12 strains with special references to the effects of SOS repair deficiencies. (1) The umuC gene is inducible by Blm and the induction is regulated by the lexA and recA genes. (2) The lexA and recA mutants are slightly more sensitive to Blm-killing than wild-type strain. (3) The plating efficiency of UV-irradiated λ-phage increased by Blm treatment of the host cell. This increase was not observed in the umuC mutant. The plating efficiency of UV-irradiated λ-phage was drastically reduced in the lexA and recA strains treated with Blm. (4) No significant increase of the reversion of nonsense mutation ( his-4 to His +) in AB1157 by the treatment of Blm was observed. Possible implications of these results are discussed.

Role of the E. coli umuC gene product in the repair of single-stranded DNA phage.

The umuC product of Escherichia coli has been suggested to have a central role in SOS induced error prone replication of DNA (Kato and Shinoura 1977). To investigate this possibility, we examined the effect of umuC mutations on error prone repair of single and double-stranded DNA phages. No Weigle reactivation of M13 phage was detected in a umuC mutant. Reactivation of lambda phage was reduced but still evident. However mutagenesis occurred in both cases. These results suggest that induced error prone replication of phage DNA can occur via umuC dependent (transdimer synthesis) and umuC independent mechanisms.

Mutagenesis and repair deficiencies of Escherichia coli umuC mutants are suppressed by the plasmid pKM101

The presence of the drug resistance plasmid pKM101 restored the ability of Escherichia coli umuC mutant strains to be mutated by methyl methanesulfonate. Inducible (Weigle) reactivation of ultraviolet-irradiated bacteriophage lambda was not observed in uvrA6 umuC mutant strains lacking pKM101 but was observed if the plasmid was present in the strains. In a uvrA+ umuC36 strain pKM101 increased the efficiency of the Weigle reactivation process. Plasmid-mediated UV-resistance and plasmid-mediated phage reactivation were observed in umuC(pKM101) strains both in uvrA+ and uvrA6 backgrounds. No restoration of methyl methanesulfonate mutability by pKM101 was observed in umuC36 recA56 strains. pKM101 mutants unable to enhance mutagenesis in umuC+ backgrounds also had no effect on methyl methanesulfonate mutagenesis in umuC mutant strains. Neither a umuC mutation nor the presence of pKM101 affected the UV induction of protein X, the recA protein. Hypotheses relating the mode of action of pKM101 to the process of mutagenesis and inducible phage reactivation are discussed.