Escherichia coli DNA helicase II (uvrD gene product) catalyzes the unwinding of DNA.RNA hybrids in vitro.

DNA helicase II is a well-characterized Escherichia coli enzyme capable of unwinding duplex DNA and known to be involved in both methyl-directed mismatch repair and excision repair of pyrimidine dimers. Here it is shown that this enzyme also catalyzes the ATP-dependent unwinding of a DNA.RNA hybrid consisting of a radioactively labeled RNA molecule annealed on M13 single-stranded DNA. The DNA.RNA unwinding reaction required less protein to unwind more base pairs than the corresponding unwinding of duplex DNA. In addition, the rate of unwinding of the DNA.RNA hybrid was more than an order of magnitude faster than unwinding of a DNA partial duplex of similar length. The unwinding of the DNA.RNA hybrid is a property unique to helicase II since helicase I, Rep protein, and helicase IV failed to catalyze the reaction. In light of these results it seems likely that helicase II is involved in some previously unrecognized aspect of nucleic acid metabolism, in addition to its known roles in DNA repair reactions.     

Cloning of the uvrC gene of Escherichia coli: expression of a DNA repair gene.

We have cloned the uvrC gene of Escherichia coli, using an F' plasmid carrying the uvrC region as a source of DNA. Two plasmids, pSC101 and pBR322, were used as cloning vectors. The recombinant plasmids were selected for their ability to complement the uvrC defect of E. coli strains AB1884 and N177. We conclude that the uvrC structural gene is contained in a 1.9-kilobase DNA fragment. The protein encoded by the uvrC gene appears to have a monomer molecular weight of 64,500 as analyzed by denaturing polyacrylamide gel electrophoresis. Strains containing multicopy uvrC+ plasmids overproduce a factor that is missing in lysates of uvrC- mutants and required for an in vitro model repair reaction. The expression of uvrC+ hybrid plasmids suggests that the structural gene is separated by at least 0.8 kilobase from the regulatory region.     

Cloning a eukaryotic DNA glycosylase repair gene by the suppression of a DNA repair defect in Escherichia coli.

If eukaryotic genes could protect bacteria with defects in DNA repair, this effect could be exploited for the isolation of eukaryotic DNA repair genes. We have thus cloned a DNA repair gene from Saccharomyces cerevisiae that directs the synthesis of a DNA glycosylase that specifically releases 3-methyladenine from alkylated DNA and in so doing protects alkylation-sensitive Escherichia coli from killing by methylating agents. The cloned yeast gene was then used to generate a mutant strain of S. cerevisiae that carries a defect in the glycosylase gene and is extremely sensitive to DNA methylation. This approach may allow the isolation of a large number of eukaryotic DNA repair genes.     

The delta subunit of Escherichia coli DNA polymerase III holoenzyme is the dnaX gene product.

The delta subunit of DNA polymerase III holoenzyme has been purified extensively with an assay for phi X174 DNA synthesis using core (pol III) and beta and gamma subunits. Either the purified delta subunit or the purified DNA polymerase III holoenzyme can complement a defective enzyme fraction from the conditional replication mutant SG133 described by Sevastopoulos et al. [Sevastopoulas, C.G., Wehr, C.T. & Glaser, D. A. (1977) Proc. Natl. Acad. Sci. USA 74, 3485-3489]. It has been established by Henson et al. [Henson, J.M., Chu, H., Irwin, C.A. & Walker, J.R. (1979) Genetics 92, 1,41-1059] that SG133 has two temperature-sensitive mutations, called dnaX and dnaY. The crude enzyme source from dnaX can be complemented by the delta subunit and by DNA polymerase III holoenzyme. By contrast, the core DNA polymerase III and the beta and gamma subunits are unable to complement this defective enzyme fraction. Thus, the delta subunit of DNA polymerase III holoenzyme appears to be the dnaX gene product of Escherichia coli.     

A second DNA methyltransferase repair enzyme in Escherichia coli.

The Escherichia coli ada-alkB operon encodes a 39-kDa protein (Ada) that is a DNA-repair methyltransferase and a 27-kDa protein (AlkB) of unknown function. By DNA blot hybridization analysis we show that the alkylation-sensitive E. coli mutant BS23 [Sedgwick, B. & Lindahl, T. (1982) J. Mol. Biol. 154, 169-175] is a deletion mutant lacking the entire ada-alkB operon. Despite the absence of the ada gene and its product, the cells contain detectable levels of a DNA-repair methyltransferase activity. We conclude that the methyltransferase in BS23 cells is the product of a gene other than ada. A similar activity was detected in extracts of an ada-10::Tn10 insertion mutant of E. coli AB1157. This DNA methyltransferase has a molecular mass of about 19 kDa and transfers the methyl groups from O6-methylguanine and O4-methylthymine in DNA, but not those from methyl phosphotriester lesions. This enzyme was not induced by low doses of alkylating agent and is expressed at low levels in ada+ and a number of ada- E. coli strains.     

Model for regulation of Escherichia coli DNA repair functions.

A feedback loop for the regulation of the rec/lex-mediated DNA repair system is proposed. This model was formulated from experiments on the genetic and metabolic regulation of the rate of synthesis of protein X performed in this laboratory, and from genetic data obtained in other laboratories. Protein X is proposed to prevent DNA degradation by the recBC-coded exonuclease. The model states tht: (1) The lex (or exrA in E. coli B) gene codes for a repressor. (2) This repressor binds to an operator region of DNA consisting of the tif-zab region at 51 minutes on the E. coli chromosome. (3) The operator region controls the production of several proteins involved in DNA repair, including protein X. (4) The recA gene product is required to remove the lex-coded repressor from the operator. Thre recA gene could code for an antirepressor (inducer protein or a protease) or a modifer of recBC nuclease action; (5) Low molecular weight products of DNA degradation are effectors that activate the system. (6) Protein X limits recBC nuclease action by binding to single-stranded DNA.     

Role of the host in virus assembly: cloning of the Escherichia coli groE gene and identification of its protein product.

Correct assembly of the heads of bacteriophages lambda and T4 requires the function of the groE gene of the Escherichia coli host. We have isolated a transducing derivative of lambda, called lambda gt-Ec.groE, that carries a functional copy of the groE gene. Unlike wild-type lambda, this phage is able to form plaques on hosts with a mutant groE gene. We have isolated an amber mutation in the groE gene carried by the phage, and this has made it possible to identify the groE product as a protein of molecular weight 65,000. In the phage, the groE gene is under the control of an early phage promoter.     

Identification of Escherichia coli DNA helicase I as the traI gene product of the F sex factor.

Active DNA helicase I (Mr 180,000) can be isolated from Escherichia coli F+ strains but not F- strains. The transfer of the F sex factor to F- strains by conjugation permits the purification of the enzyme from the transconjugant strains. We conclude from this that helicase I is coded for by a portion of the F factor. Results also obtained by using recombinant plasmids carrying different DNA fragments of the F factor transfer region suggest that DNA helicase I is identical to the product of traI, one of the transfer genes of the F factor.     

Identification of the lexA gene product of Escherichia coli K-12.

The Escherichia coli lexA gene encodes a product important in induction of the recA gene and the expression of various cellular functions, including mutagenesis and prophage induction. As a start in a biochemical analysis of the lexA function, a family of lambda transducing phages carrying lexA+, lexA3, lexA3 spr-54, and lexA3 spr-55 alleles of the lexA gene was isolated and characterized. Polypeptides synthesized by these phages were examined. lambdalexA+ made a distinctive protein 24 kilodaltons (kd) in size. Lambda lexA3, which encodes an active mutant form of the protein dominant to wild-type function, made a slightly larger protein 25 kd in size. The latter protein was shown to be the mutant lexA3 gene product by the fact that lambda lexA3 spr-55, which carries an amber mutation in lexA3, made the 25-kd protein in hosts with an amber suppressor but not in a suppressor-free host. In hosts carrying a multicopy lexA3 plasmid, neither the 25-kd nor the 24-kd protein was made. This result suggests that lexA is autoregulated and that expression of the 24-kd protein made by lambda lexA+ is subject to the same controls. This and other evidence argues that the 24-kd protein is the product of the wild-type lexA+ gene.     

Escherichia coli recA gene product inactivates phage lambda repressor.

Phage lambda repressor is inactivated and cleaved into two detectable fragments during incubation with purified Escherichia coli recA gene protein in vitro, in a reaction that requires ATP. This reaction reproduces the recA-dependent inactivation of repressor that occurs in vivo during induction of the SOS functions. The proteolytic activity may reside in the recA protein itself and may be a fundamental activity of it.     

The identification of the mot gene product with Escherichia coli-lambda hybrids.

Molecular cloning techniques were used to construct lambda-E. coli hybrid bacteriophage carrying genes involved in bacterial flagellar motility (mot) and chemotaxis (cheA). A series of hybrid bacteriophage without each of these genes was also prepared.When paralyzed mutants of E. coli were infected with lambda that carried the mot gene, the ability of the bacterium to swim was rapidly restored. The restoration of motility was the result of the synthesis and insertion into the cell membrane of a protein with an apparent molecular weight of 31,000 (the Mot protein). Another polypeptide with a mobility on acrylamide gel electrophoresis which corresponded to a molecular weight of 39,000 was associated with the cheA gene. The presence of this polypeptide alone was not sufficient to restore chemotactic activity to mutant cheA strains. It was suggested that only a portion of the cheA gene was cloned, and thus the 39,000 protein may be a partial product of the cheA gene, or the product of a second mot gene.     

Chromosomal directionality of DNA mismatch repair in Escherichia coli

Defects in DNA mismatch repair (MMR) result in elevated mutagenesis and in cancer predisposition. This disease burden arises because MMR is required to correct errors made in the copying of DNA. MMR is bidirectional at the level of DNA strand polarity as it operates equally well in the 5′ to 3′ and the 3′ to 5′ directions. However, the directionality of MMR with respect to the chromosome, which comprises parental DNA strands of opposite polarity, has been unknown. Here, we show that MMR in Escherichia coli is unidirectional with respect to the chromosome. Our data demonstrate that, following the recognition of a 3-bp insertion-deletion loop mismatch, the MMR machinery searches for the first hemimethylated GATC site located on its origin-distal side, toward the replication fork, and that resection then proceeds back toward the mismatch and away from the replication fork. This study provides support for a tight coupling between MMR and DNA replication.DNA can be mutated following damage caused by exposure to chemical or physical mutagens or by errors in DNA metabolism, during replication, recombination, or repair (1). Although replicative DNA polymerases have proofreading activities, they cannot avoid a low frequency of incorporation of noncomplementary deoxynucleoside triphosphates (dNTPs). It has been estimated that there are 10⁻⁵–10⁻⁶ misincorporation events per replicated base pair (2). Uncorrected errors of this kind (known as mismatches) will be converted into mutations, which have the potential to perturb biological processes in the next round of DNA replication. The DNA mismatch repair (MMR) system is a key DNA guardian that ensures the removal of the misincorporated nucleotides and thereby maintains genomic integrity. The MMR system has a defined substrate range, with different efficiencies of correction of single base mismatches and a 3–4 base size limit for the correction of insertion/deletion loops (IDLs) (3–6). This system is conserved in almost all organisms, with the exception of most Actinobacteria and Mollicutes, and parts of the archaea (7).When the replication machinery inserts a noncomplementary dNTP in the nascent strand of the E. coli chromosome, the MutS protein binds to the mismatch and recruits the MutL protein (8). These two proteins activate the endonuclease MutH, which nicks the unmethylated strand of a hemimethylated GATC site to initiate removal of the nascent strand containing the mismatch (9, 10). Following the passage of the replisome, GATC motifs remain transiently hemimethylated before methylation of the nascent strand by the Dam methyltransferase enzyme and can therefore be used to distinguish between parental and nascent strands (11, 12). In vitro, MutH can distinguish between these strands by using hemimethylated GATC motifs located within a 2-kb distance of the mismatch, either on the 3′ or the 5′ side (13, 14). UvrD helicase uses the incision made by MutH at the hemimethylated GATC motif as an entry point to unwind the nascent strand. One or more of the four exonucleases (ExoI, ExoVII, RecJ, and ExoX), depending on the required polarity of degradation, resects the unwound nascent strand (15, 16). The single-stranded parental DNA is immediately bound by the single-strand DNA-binding protein (SSB) (17). The DNA polymerase III holoenzyme then correctly resynthesizes the nascent strand and DNA ligase seals the remaining nick (16, 18).Most of the previous studies of MMR have been based on in vitro experiments using linear or closed-circular heteroduplex DNA substrates in defined systems (19–21). These in vitro studies have shown that an incision of the nascent strand can occur either on the 3′ or the 5′ side of a mismatch, depending on the position of the hemimethylated GATC motif recognized by MutH (22, 23). Using electron microscopy and end labeling, Grilley et al. have shown that the single-stranded DNA region created after an excision reaction lies between the mismatch and the closest GATC motif (15). A hemimethylated GATC motif recognized on the 3′ side of the mismatch requires a 3′ to 5′ exonuclease (e.g., ExoI or ExoX), and ExoVII or RecJ cleaves the single-stranded DNA when a hemimethylated GATC motif is recognized on the 5′ side of the mismatch (5′ to 3′ cleavage).Therefore, in vitro, the cleavage reaction of the MMR system is bidirectional (15, 16). However, in vivo, an MMR system that is bidirectional with respect to DNA polarities could nevertheless be unidirectional with respect to the chromosome. In fact, a unique directionality of MMR with respect to DNA replication would explain the evolution of bidirectionality at the level of strand polarities as mismatches on both the leading and lagging strands need to be repaired. Fig. 1A distinguishes directionality at the level of strand polarity from directionality at the level of the chromosome and illustrates how directionality relative to the replication fork can lead to bidirectionality of resection polarities. Blackwood and collaborators found that MMR at the site of an unstable trinucleotide repeat (TNR) array caused a stimulation of recombination at a nearby 275-bp tandem repeat. This stimulation occurred only when the tandem repeat was placed on the origin-proximal side the TNR that had generated a high frequency substrate for MMR (24). This result suggested that MMR might be directional in a chromosomal context. We have now tested this hypothesis and shown that the MMR system of E. coli has a unique chromosomal directionality.Open in a separate windowFig. 1.Directionality of MMR. (A) Visualization of MMR in the context of DNA replication. The two possible directionalities of MMR with respect to the chromosome are depicted in the context of the replication fork where mismatches arise. The MMR complex has the potential to scan the chromosome for a hemimethylated GATC site in the direction of movement of the replication fork (i) or in the opposite direction, away from the replication fork (ii). Mismatches are shown on both the leading and lagging strands. However, in reality they are most likely to be present in either one or other of these two locations at a given time. It can be seen that if the scanning for a hemimethylated GATC is unidirectional with respect to the chromosome because of the direction of movement of the replication fork, this can lead to bidirectional resection to repair mismatches on both the leading and the lagging strands. (B) Schematic representation of the locus of interest and the experimental design to investigate the influence of GATC sites on MMR and tandem repeat recombination associated with MMR. The native GATC motifs within the 5-kb region surrounding the CTG⋅CAG TNR array are shown. There are thirteen GATC motifs within the 2.5-kb region on the origin-proximal side of the TNR, of which, two (P128 and P224) are situated between the TNR and the site of insertion of the zeocin cassette. On the origin-distal side of the TNR, there are nine GATC motifs in the first 2.5-kb region. The E. coli chromosome is shown as a horizontal blue cylinder, the TNR is shown as a green band and the zeocin cassette is shown as two tandem red cylinders. GATC motifs are shown by amber bands with their respective names based on their positions. “P” and “D” correspond to the origin-proximal side and the origin-distal side of the TNR, respectively, whereas the number indicates the distance between the GATC motif and the TNR.     

Protein X is the product of the recA gene of Escherichia coli.

The inducible protein X of Escherichia coli has been compared to the recA+ protein made by specialized recA transducing phages. The molecular weights and isoelectric points of these proteins are identical. Two mutations located in the recA gene that alter the electrophoretic mobility or the isoelectric point of protein X have been studied. A recA12 mutant strain, deficient in homologous recombination and repair, produces a smaller-than-normal protein X. A transducing phage carrying the recA12 allele directs the synthesis of a smaller recA protein after infection of irradiated cells. A transducing phage carrying the recA region of a tif-1 mutant strain codes for a recA protein with an isoelectric point more basic than that of the lambdaprecA+ product. The protein X of a tif-1 mutant strain shows an identical shift in its isoelectric properties. Examination of several tsl- recA- strains indicates that protein X can be induced in several missense recA mutants but is not detected in tsl- strains carrying amber or deletion mutations of the recA gene. These results demonstrate that protein X is the product of the recA gene and that the tif-1 mutation alters the properties of the recA protein. A model is suggested for autoregulation of the recA protein in the induction of functions expressed in response to DNA damage (SOS functions).     

Identification of the epsilon-subunit of Escherichia coli DNA polymerase III holoenzyme as the dnaQ gene product: a fidelity subunit for DNA replication.

Based on extensive genetic and biochemical studies, the multisubunit DNA polymerase III holoenzyme is considered responsible for the chain-elongation stage in replication of the genome of Escherichia coli and is thus expected to be the major determinant of fidelity as well. Previous experiments have shown that two mutations conferring a very high mutation rate on E. coli, mutD5 and dnaQ49, decrease severely the 3' leads to 5' exonucleolytic editing activity of the polymerase III holoenzyme. To identify more precisely the nature of these mutations, we have carried out genetic mapping and complementation experiments. From these studies and experiments by others, we conclude that the most potent general mutator mutations in E. coli occur in a single gene, dnaQ. To define further the role of the dnaQ gene, we have used two-dimensional gel electrophoresis to compare the labeled dnaQ gene product with purified polymerase III holoenzyme. The dnaQ product comigrates with the epsilon-subunit, a 25-kilodalton protein of the polymerase III "core" enzyme. We conclude that the epsilon-subunit of polymerase III holoenzyme has a special role in defining the accuracy of DNA replication, probably through control of the 3' leads to 5' exonuclease activity.     

Cell sensitivity to irradiation and DNA repair processes. I. DNA damage and its repair in Escherichia coli

Basic types of DNA damage produced by gamma-radiation in cells are reviewed. Different DNA injuries are related to various levels of DNA repair processes, established in the case of Escherichia coli cells. The role of the balance of repair enzyme activities is considered in connection with the induction of enzymatic DNA double-strand breaks (DSBs). The concept of "metastable sites" has been introduced. "Metastable sites" are formed from great nucleolytic gaps. They are measured as DSBs although they can be repaired as single-strand breaks (SSBs). A simple mathematical model of the inactivation of different mutants of E. coli cells has been constructed on the basis of available experimental data. Kinetic equations of the model have been solved and some parameters estimated for both sensitive and resistant mutants.     

Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product.

In previous studies, Holliday junctions generated during RecA-mediated strand-exchange reactions were resolved by fractionated Escherichia coli extracts. We now report the specific binding and cleavage of synthetic Holliday junctions (50 base pairs long) by a fraction purified by chromatography on DEAE-cellulose, phosphocellulose, and single-stranded DNA-cellulose. The cleavage reaction provided a sensitive assay with which to screen extracts prepared from recombination/repair-deficient mutants. Cells with mutations in ruvC lack the nuclease activity that cleaves synthetic Holliday junctions in vitro. This deficiency was restored by a multicopy plasmid carrying a ruvC+ gene that overexpressed junction-resolving activity. The UV sensitivity and deficiency in recombinational repair of DNA exhibited by ruv mutants lead us to suggest that RuvC resolves Holliday junctions in vivo.     

Genetic identification of the DNA binding domain of Escherichia coli LexA protein.

Two genetic approaches were taken to define the DNA binding domain of LexA protein, the repressor of the Escherichia coli SOS regulon. First, several dominant negative lexA mutants defective in DNA binding were isolated. The mutations altered amino acids in a region similar to the helix-turn-helix, a DNA binding domain of other repressors and DNA binding proteins. Second, the region encoding the predicted DNA recognition helix was subjected to oligonucleotide-directed mutagenesis and mutant LexA proteins with altered or relaxed specificity for several recA operator positions were isolated. By examining the effects of a series of amino acid substitutions on repressor specificity, it was shown that a glutamic acid residue at position 45 in LexA protein is important for recognition of the first base pair (G.C) in the recA operator.     

Mechanisms of mutagenesis in the Escherichia coli mutator mutD5: role of DNA mismatch repair.

To investigate the mechanisms of spontaneous mutation in the Escherichia coli mutD5 mutator strain, 502 mutations generated in this strain in the N-terminal part of the lacI gene were sequenced (i-d mutations). Since the mutator strength of this strain depends on the medium in which it grows, mutations were analyzed in both minimal medium (moderate mutator activity) and rich medium (high mutator activity). In either case, 95% of all mutations were base substitutions and 5% were single-base deletions. However, the nature and site distribution of the base substitutions differed dramatically for the two conditions. In minimal medium (mutation frequency, 480-fold above background), a majority (62%) were transversions, notably A.T----T.A at three 5'-GTGG-3' sequences. Most (64%) of the transitions under this condition occurred at specific sequences that are suggestive of a "dislocation" type of mutagenesis. In rich medium (mutation frequency, 37,000-fold above background), 90% of the base substitutions were transitions. These observations suggest that different modes of mutagenesis operate under the two conditions. mutD5 cells have been reported to be defective in exonucleolytic proofreading during DNA replication. The present data suggest that mutD cells in rich medium also suffer a defect in mutHLS-encoded mismatch correction. This hypothesis was confirmed by the direct measurement of mismatch repair in mutD5 cells by transfection of M13mp2 heteroduplex DNA.     

Involvement of helicase II (uvrD gene product) and DNA polymerase I in excision mediated by the uvrABC protein complex.

The bimodal-incision nature of the reaction of UV-irradiated DNA catalyzed by the Escherichia coli uvrABC protein complex potentially leads to excision of a 12- to 13-nucleotide-long damaged fragment. However, the oligonucleotide fragment containing the UV-induced pyrimidine dimer is not released under nondenaturing in vitro reaction conditions. Also, the uvrABC proteins are stably bound to the incised DNA and do not turn over after the incision event. In this communication it is shown that release of the damaged fragment from the parental uvrABC-incised DNA is dependent upon either chelating conditions or the simultaneous addition of the uvrD gene product (helicase II) and the polA gene product (DNA polymerase I) when polymerization of deoxynucleoside triphosphate substrates is concomitantly catalyzed. The product of this multiprotein-catalyzed series of reactions serves as a substrate for polynucleotide ligase, resulting in the restoration of the integrity of the strands of DNA. The addition of the uvrD protein to the incised DNA-uvrABC complex also results in turnover of the uvrC protein. It is suggested that the repair processes of incision, excision, resynthesis, and ligation are coordinately catalyzed by a complex of proteins in a "repairosome" configuration.