Biomedical subjects

R Woodgate

Publications and source records attributed to R Woodgate.

90 records · Page 5Linked to original sources

PubMed1992

Activity of the purified mutagenesis proteins UmuC, UmuD', and RecA in replicative bypass of an abasic DNA lesion by DNA polymerase III.

The introduction of a replication-inhibiting lesion into the DNA of Escherichia coli generates the induced, multigene SOS response. One component of the SOS response is a marked increase in mutation rate, dependent on RecA protein and the induced mutagenesis proteins UmuC and UmuD. A variety of previous indirect approaches have indicated that SOS mutagenesis results from replicative bypass of the DNA lesion by DNA polymerase III (pol III) holoenzyme in a reaction mediated by RecA, UmuC, and a processed form of UmuD termed UmuD'. To study the biochemistry of SOS mutagenesis, we have reconstituted replicative bypass with a defined in vitro system containing purified protein and a DNA substrate with a single abasic DNA lesion. The replicative bypass reaction requires pol III, UmuC, UmuD', and RecA. The nonprocessed UmuD protein does not replace UmuD' but inhibits the bypass activity of UmuD', perhaps by sequestering UmuD' in a heterodimer. Our experiments demonstrate directly that the UmuC-UmuD' complex and RecA act to rescue an otherwise stalled pol III holoenzyme at a replication-blocking DNA lesion.

Bacterial Proteins↗

PubMed1992

Escherichia coli umuDC mutants: DNA sequence alterations and UmuD cleavage.

The products of the chromosomally encoded umuDC genes are directly required for mutagenesis in Escherichia coli. Strains with either umuD or umuC mutations are rendered phenotypically non-mutable. To ascertain the molecular basis of this non-mutability, we determined the DNA sequence alterations of seven chromosomal umuDC mutants. Six mutants (umuD1, umuD44, umuD77, umuC36, umuC25, and umuC104) were found to be single base-pair substitutions that resulted in missense mutations. The Tn5 transposon insertion mutation (umuC122) resulted in a missense mutation followed immediately by a termination codon, producing a truncated UmuC protein lacking 102 carboxyl-terminal amino acids. All of the mutations were found to reside in regions of the UmuD and UmuC proteins that share high homology with analogous proteins. Chemiluminescent immunoassays revealed that the umuD1, umuD44, and umuD77 mutations all resulted in a non-cleavable UmuD protein. Because UmuD cleavage is a prerequisite for mutagenesis, the lack of UmuD processing appears to be the molecular basis for the non-mutable phenotype in these strains. These studies re-emphasize the critical nature of the RecA-mediated cleavage of UmuD for inducible mutagenesis and provide insights into the functional domains of the UmuC protein.

Bacterial Proteins↗

PubMed1992

Construction of a umuDC operon substitution mutation in Escherichia coli.

Using a specialized transducing lambda phage, the umuDC operon of Escherichia coli was deleted and replaced with the chloramphenicol acetyltransferase gene. The delta (umuDC)595::cat mutation was subsequently transferred by generalized P1 transduction into a variety of genetic backgrounds. It is concluded that the UmuDC proteins, which are normally required for inducible mutagenesis, are not essential for cell survival.

Bacteriophage lambda↗

PubMed1992

Mutagenesis induced by bacterial UmuDC proteins and their plasmid homologues.

The popular image of a world full of pollutants mutating DNA is only partly true since there are relatively few agents which can subtly and directly change base coding; for example, some alkylating agents alter guanine so that it pairs like adenine. Many more mutagens are less subtle and simply destroy coding altogether rather than changing it. Such mutagens include ultraviolet light, X-rays, DNA cross-linkers and other agents which make DNA breaks or large adducts. In Escherichia coli, mutagenesis by these agents occurs during a DNA repair process which increases cell survival but with an inherent possibility of changing the original sequence. Such mutagenic DNA repair is, in part, encoded by the E. coli umuDC operon. This article reviews the structure, function, regulation and evolution of the umuDC operon and similar genes found both in other species and on naturally occurring plasmids.

Amino Acid Sequence↗

PubMed1992

The enhanced mutagenic potential of the MucAB proteins correlates with the highly efficient processing of the MucA protein.

Inducible mutagenesis in Escherichia coli requires the direct action of the chromosomally encoded UmuDC proteins or functional homologs found on certain naturally occurring plasmids. Although structurally similar, the five umu-like operons that have been characterized at the molecular level vary in their ability to enhance cellular and phage mutagenesis; of these operons, the mucAB genes from the N-group plasmid pKM101 are the most efficient at promoting mutagenesis. During the mutagenic process, UmuD is posttranslationally processed to an active form, UmuD'. To explain the more potent mutagenic efficiency of mucAB compared with that of umuDC it has been suggested that unlike UmuD, intact MucA is functional for mutagenesis. To examine this possibility, we have overproduced and purified the MucA protein. Although functionally similar to UmuD, MucA was cleaved much more rapidly both in vitro and in vivo than UmuD. In vivo, restoration of mutagenesis functions to normally nonmutable recA430, recA433, recA435, or recA730 delta(umuDC)595::cat strains by either MucA+ or mutant MucA protein correlated with the appearance of the cleavage product, MucA'. These results suggest that most of the differences in mutagenic phenotype exhibited by MucAB and UmuDC correlate with the efficiency of posttranslational processing of MucA and UmuD rather than an inherent activity of the unprocessed proteins.

Bacterial Proteins↗

PubMed1991

Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage.

Most of the inducible mutagenesis observed in Escherichia coli after treatment with many DNA damaging agents is dependent upon the products of the umuD,C operon. RecA-mediated proteolytic processing of UmuD yields a carboxyl-terminal fragment (UmuD') that is active for mutagenesis. Processing of UmuD is therefore a critical step in the fixation of mutations. In this paper we have analyzed the requirements for UmuD processing in vivo. Standard immuno-detection assays, coupled with a sensitive chemiluminescence detection assay, have been utilized to probe levels of chromosomally encoded Umu proteins from whole-cell E. coli extracts. We found that the derepression of additional SOS gene products, other than RecA, was not required for UmuD processing. Moreover, efficient cleavage of UmuD was observed only in the presence of elevated levels of activated RecA, suggesting that efficient processing would occur only under conditions of severe DNA damage. Detection of chromosomally encoded Umu proteins has allowed us, for the first time, to measure directly the cellular steady-state levels of these proteins under various SOS inducing conditions. UmuD was present at approximately 180 copies per uninduced cell and was measured at approximately 2400 copies per cell in strains that lacked a functional repressor. Induced levels of UmuC were approximately 12-fold lower than UmuD with approximately 200 molecules per cell. These levels of cellular UmuC protein suggest that it functions through specific protein-DNA or protein-protein interactions, possibly as a lesion recognition protein or by interacting with DNA polymerase III.

Bacterial Proteins↗

PubMed1991

Induction and cleavage of Salmonella typhimurium UmuD protein.

SOS mutagenesis in prokaryotes is dependent upon the inducible activity of the chromosomally encoded UmuDC proteins, or homologous proteins such as MucAB or ImpCAB which are found on naturally occurring plasmids. Relative to Escherichia coli, however, Salmonella typhimurium is much less responsive to the mutagenic effects of DNA-damaging agents, despite the fact that it possesses both chromosomally and plasmid encoded umu-like operons. In E. coli, activation of the UmuD mutagenesis protein to UmuD' via RecA-mediated proteolysis is a critical step in the mutation fixation pathway. We have used a polyclonal antiserum raised against the E. coli UmuD and UmuD' proteins to show that S. typhimurium expresses cross-reacting material only after treatment with the DNA-damaging agent mitomycin C. The S. typhimurium umuDC operon, therefore, appears to be regulated by mechanisms similar to the E. coli umuDC operon. After induction, the S. typhimurium UmuD protein was processed to UmuD' in both S. typhimurium and E. coli. However, the S. typhimurium UmuD protein appears to be cleaved more efficiently than the E. coli UmuD protein under similar conditions. The data suggest that conversion of UmuD to the mutagenically active UmuD' is not the rate-limiting factor accounting for the weakly mutable phenotype of S. typhimurium.

Bacterial Proteins↗

PubMed1991

Mutagenic DNA repair in enterobacteria.

Sixteen species of enterobacteria have been screened for mutagenic DNA repair activity. In Escherichia coli, mutagenic DNA repair is encoded by the umuDC operon. Synthesis of UmuD and UmuC proteins is induced as part of the SOS response to DNA damage, and after induction, the UmuD protein undergoes an autocatalytic cleavage to produce the carboxy-terminal UmuD' fragment needed for induced mutagenesis. The presence of a similar system in other species was examined by using a combined approach of inducible-mutagenesis assays, cross-reactivity to E. coli UmuD and UmuD' antibodies to test for induction and cleavage of UmuD-like proteins, and hybridization with E. coli and Salmonella typhimurium umu DNA probes to map umu-like genes. The results indicate a more widespread distribution of mutagenic DNA repair in other species than was previously thought. They also show that umu loci can be more complex in other species than in E. coli. Differences in UV-induced mutability of more than 200-fold were seen between different species of enteric bacteria and even between multiple natural isolates of E. coli, and yet some of the species which display a poorly mutable phenotype still have umu-like genes and proteins. It is suggested that umDC genes can be curtailed in their mutagenic activities but that they may still participate in some other, unknown process which provides the continued stimulus for their retention.

Blotting, Southern↗

PubMed1989

Non-mutability by ultraviolet light in uvrD recB derivatives of Escherichia coli WP2 uvrA is due to inhibition of RecA protein activation.

The deficiency in UV mutagenesis in uvrD3 recB21 strains of E. coli is almost completely overcome by constitutive activation of RecA protein and expression of the SOS system (by recA730 or 43 degrees C treated recA441 lexA71). When SOS was expressed but RecA protein not self-activated (recA441 lexA71 at 30 degrees C), uvrD3 recB21 still reduced UV mutagenesis at low doses. The uvrD3 recB21 combination is therefore inhibiting activation of RecA protein. It is suggested that the DNA unwinding activity of the products of the uvrD and recB genes may be involved in generating single-stranded DNA needed to activate RecA protein both for the cleavage of LexA repressor and for a further role in UV mutagenesis.

DNA Repair↗

PubMed1989

UmuC mutagenesis protein of Escherichia coli: purification and interaction with UmuD and UmuD'.

The introduction of a replication-inhibiting lesion into the DNA of Escherichia coli produces a marked elevation in mutation rate. The mutation pathway is a component of the induced, multigene SOS response. SOS mutagenesis is a tightly regulated process dependent on two RecA-mediated proteolytic events: cleavage of the LexA repressor to induce the UmuC and UmuD mutagenesis proteins, and cleavage of UmuD to UmuD' to activate the mutation pathway. To investigate the protein-protein interactions responsible for SOS mutagenesis, we have studied the interaction of UmuC, UmuD, and UmuD'. To probe intracellular interaction, we have used immunoprecipitation techniques with antibodies against UmuC or UmuD and UmuD'. We have found that antibody to UmuC precipitates UmuD' from cell extracts, and antibody to UmuD and UmuD' precipitates UmuC. Thus we conclude that UmuC probably associates tightly with UmuD' in cells. For biochemical studies, we have purified the UmuC and UmuD' proteins to use with the previously purified UmuD. UmuC associates strongly with an affinity column of UmuD and UmuD', eluting only under strongly dissociating conditions (2 M urea or 1.5 M KSCN). UmuC also associates efficiently with UmuD or UmuD' in solution, as judged by velocity sedimentation in a glycerol gradient. The likely stoichiometry is one UmuC with a dimeric UmuD or UmuD'. From these experiments and previous work, we infer that SOS mutagenesis depends on the action of the UmuC-UmuD' complex and probably RecA to rescue a stalled DNA polymerase III holoenzyme at the DNA lesion.

Bacterial Proteins↗

PubMed1988

UmuD mutagenesis protein of Escherichia coli: overproduction, purification, and cleavage by RecA.

The mutation rate of Escherichia coli increases approximately 100-fold after treatment with replication-inhibiting agents such as UV light. This enhanced mutation rate requires the action of the UmuD and UmuC proteins, which are induced as part of the SOS response to DNA damage. To initiate a biochemical characterization of the role of these proteins, we have developed a plasmid system that gives efficient expression of the umuD and umuC genes. The umuD and umuC genes were placed under the control of a regulated phage lambda PL promoter and a synthetic ribosome-binding site, and the distance to the UmuD start was adjusted to maximize gene expression. Starting from this overproduction system, we have purified the UmuD protein and studied its interaction with RecA. The SOS response is turned on by the capacity of RecA protein to mediate cleavage of the LexA repressor for SOS-controlled operons. Others have shown that UmuD exhibits sequence homology to LexA around the cleavage site, suggesting a possible cleavage reaction for UmuD. We show that RecA mediates cleavage of UmuD, probably at this site. As with LexA, UmuD also undergoes a self-cleavage reaction. We infer that RecA-mediated cleavage of UmuD is another role for RecA in SOS mutagenesis, probably activating UmuD for its mutagenic function.

Bacterial Proteins↗

PubMed1987

Current understanding of UV-induced base pair substitution mutation in E. coli with particular reference to the DNA polymerase III complex.

UV mutagenesis in E. coli is believed to occur in two discrete steps. The second step involves continued DNA synthesis beyond a blocking lesion in the template strand. This bypass step requires induced levels of umuD and umuC gene products and activated recA protein. DNA polymerase III may be involved since a dnaE mutator strain (believed to have defective base selection) is associated with enhanced UV mutagenesis in conjunction with a genetic background permitting the bypass step. In non-UV-mutable umu and lexA strains, UV mutagenesis can be demonstrated if delayed photoreversal is given. This is interpreted as indicating that an earlier misincorporation step can occur in such strains but the resulting mutations do not survive because the bypass step is blocked. The misincorporation step does not require any induced SOS gene products and can occur either at the replication fork or during repair replication following excision of a DNA lesion. Neither a dnaE mutator gene (leading to a defective alpha subunit of DNA polymerase III holoenzyme) nor a mutD5 mutator gene (leading to a defective epsilon proofreading subunit) had any effect on the misincorporation step. Although this is consistent with DNA polymerase III holoenzyme not being involved in the misincorporation step, other interpretations involving the inhibition of epsilon proofreading activity by recA protein are possible. In vitro studies are reported in which sites of termination of synthesis by DNA polymerase III holoenzyme on UV-irradiated M13 mp8 DNA were examined in the presence of inhibitors of the 3'-5' proofreading exonuclease (including recA protein). No evidence was found for incorporation of bases opposite photoproducts suggesting that either inhibition is more complete in the cell and/or that other factors are involved in the misincorporation step.

DNA Polymerase III↗

PubMed1987

Mutagenic DNA repair in Escherichia coli. XIII. Proofreading exonuclease of DNA polymerase III holoenzyme is not operational during UV mutagenesis.

We have introduced a mutD5 mutation (which results in defective 3'-5'-exonuclease activity of the epsilon proofreading subunit of DNA polymerase III holoenzyme) into excision-defective Escherichia coli strains with varying SOS responses to UV light. MutD5 increased the spontaneous mutation frequency in all strains tested, including recA430, umuC122::Tn5, and umuC36 derivatives. It had no effect on UV mutability or immutability in any strain or on misincorporation revealed by delayed photoreversal in UV-irradiated umuC36, umuC122::Tn5, or recA430 bacteria. It is concluded that the epsilon proofreading subunit of DNA polymerase III holoenzyme is excluded, inhibited, or inoperative during misincorporation and mutagenesis after UV.

Arginine↗

PubMed1986

New role for photoreversible pyrimidine dimers in induction of prototrophic mutations in excision-deficient Escherichia coli by UV light.

UV mutagenesis to His+ in certain recA441 lexA51 bacteria was not photoreversible, indicating that pyrimidine dimers are not target lesions. Photoreversibility was observed in recA+ lexA51 bacteria, showing that pyrimidine dimers are needed to activate the recA+ protein (unlike the recA441 protein) to perform a function in UV mutagenesis distinct from cleavage of the lexA repressor.

DNA Repair↗

PubMed1985

Mutagenic repair in Escherichia coli: products of the recA gene and of the umuD and umuC genes act at different steps in UV-induced mutagenesis.

When excision-deficient Escherichia coli carrying umuC or umuD alleles were exposed to visible light several hours after ultraviolet irradiation, base-pair-substitution mutations were induced in these normally non-UV-mutable bacteria. It is argued that delayed photoreversal of pyrimidine dimers removes blocks to DNA replication and allows the "survival" and expression of misincorporated bases. A model for UV mutagenesis is proposed with two steps: (i) misincorporation opposite a photoproduct, which can be mediated directly by RecA protein, and (ii) bypass, only the latter process requiring umuD+ and umuC+ alleles. Basal levels of gene products are sufficient for at least some misincorporation events, although induced levels of umuD and umuC gene products are necessary for the bypass step. umuC bacteria containing the recA441 allele showed a greater yield of mutants, and those containing recA430 a reduced yield, following delayed photoreversal. The lexA51 allele (which results in constitutive derepression of RecA protein production) did not significantly alter the yield of mutants but caused them to appear marginally sooner in a recA441 umuC strain. These results emphasize that the nature of the RecA protein and not its concentration is paramount in determining the level of misincorporation. Experiments with recA441 umuC bacteria at 43 degrees C and 30 degrees C suggest that the misincorporation effect is unlikely to be attributable to cleavage of a DNA binding protein such as a repressor or a component of the polymerase complex. Moreover, misincorporation seems to occur without the need for induced synthesis of any other protein under recA control.

Bacterial Proteins↗

PubMed1984

Mutagenic repair in Escherichia coli. X. The umuC gene product may be required for replication past pyrimidine dimers but not for the coding error in UV-mutagenesis.

Bacteria of strain TK610 uvrA-6 his-4 umuC-36, when allowed to replicate their DNA for some hours after irradiation show induction of His+ mutations when subsequently exposed to visible light. It is suggested that base pair errors can be made opposite sites of pyrimidine dimers without involvement of umuC gene product but that the latter is required for continued replication past the dimer-mismatch region. Removal of the pyrimidine dimer by photoreversal allows replication to continue thus fixing the mismatched base as as mutation.

Bacterial Proteins↗

PubMedDate not supplied

The two-step model of bacterial UV mutagenesis.

Recent results are discussed which have led to a two-step model for UV mutagenesis in excision-deficient Escherichia coli. After exposure to UV, the replication fork is assumed to continue until immediately before certain photoproducts where it stops and leaves a gap which cannot be dealt with by recombination repair. In the first (misincorporation) step, bases (a proportion of which are 'wrong') are postulated to be inserted opposite the photoproduct under the direct influence of the recA gene product. These misincorporated bases can be revealed as mutations by delayed photoreversal in umuD,C and lexA (ind-) bacteria. Their level is determined by the particular allele of recA that is present (recA441 greater than recA+ greater than recA430) and their rate of formation by the amount of recA protein in the cell and the degree of enrichment of the medium. No other protein needs to be synthesized for this step to occur. The second (bypass) step requires induced levels of the products of the umuD and C genes which are postulated to facilitate continued DNA synthesis on the priming end opposite the photoproduct. In principle, further errors could be made at this stage which might appear as 'hitch-hiking' rather than 'targeted' mutations.

Bacteria↗

PubMedDate not supplied

Mutagenic DNA repair in Escherichia coli, XX. Overproduction of UmuD' protein results in suppression of the umuC36 mutation in excision defective bacteria.

Overproduction of Umu+ or UmuD' protein by means of a gene carried on a multicopy plasmid suppressed the umuC36 phenotype and permitted induction of mutations by ultraviolet light. The umuC122::Tn5 phenotype was not suppressed. Suppression of the umuC36 phenotype was only seen when excision repair was blocked by acriflavine or by an uvrA or uvrB mutation. Cleavage of UmuD to UmuD' in SOS-induced cells was not dependent upon the presence of UmuC protein. The results are interpreted in terms of a revised model in which UmuC protein is envisaged as guiding UmuD' to RecA protein which has recognized and become bound to an appropriate DNA lesion. It is suggested that the umuC36 mutation gives rise to a protein with reduced affinity for UmuD' and that the effect of this can be compensated by an excess of UmuD'.

Bacterial Proteins↗