Abstract: In many eukaryotic cells, the ribosomal RNA gene (rDNA) is composed of a highly repetitive structure. Previously, we reported the isolation of condensin mutants of Saccharomyces cerevisiae that were defective in carrying long rDNA repeat due to the loss of the replication fork barrier (RFB) protein Fob1p; thus the repeat in the mutants were dramatically contracted. The reintroduction of the FOB1 gene suppressed the contraction of the repeat. It was found that condensin mainly localized at the RFB site in a FOB1-dependent fashion. Here, we show that RNA polymerase I transcription interferes with condensin association with 35S rRNA coding regions in fob1 cells and causes dramatic contraction of rDNA repeat in the fob1 condensin double mutant. Inactivation of RNA polymerase I suppresses the dramatic contraction of the rDNA repeat in the fob1 condensin double mutant. These results suggest that association of condensin with the RFB site outside the active transcription region avoids the dramatic contraction of the rDNA repeat. We also found that the stimulation of RNA polymerase II transcription within the rDNA repeat decreased condensin association with actively transcribed regions. Thus, a characteristic of condensin is that its association with the chromatin is interfered by transcription.
Abstract: Chromosomes in eukaryotes are linear, whereas those of most, but not all, prokaryotes are circular. To explore the effects of possessing a linear genome on prokaryotic cells, we linearized the Escherichia coli genome using the lysogenic lambda-like phage N15. Linear genome E. coli were viable and their genome structure was stable. There were no appreciable differences between cells with linear or circular genomes in growth rates, cell and nucleoid morphologies, genome-wide gene expression (with a few exceptions), and DNA gyrase- and topoisomerase IV-dependent growth. However, under dif-defective conditions, only cells with a circular genome developed an abnormal phenotype. Microscopy indicated that the ends of the linear genome, but not the circular genome, were separated and located at each end of a new-born cell. When tos - the cis-element required for linearization - was inserted into different chromosomal sites, those strains with the genome termini that were more remote from dif showed greater growth deficiencies.
Abstract: An average of 200 copies of the rRNA gene (rDNA) is clustered in a long tandem array in Saccharomyces cerevisiae. FOB1 is known to be required for expansion/contraction of the repeats by stimulating recombination, thereby contributing to the maintenance of the average copy number. In Deltafob1 cells, the repeats are still maintained without any fluctuation in the copy number, suggesting that another, unknown system acts to prevent repeat contraction. Here, we show that condensin acts together with FOB1 in a functionally complemented fashion to maintain the long tandem repeats. Six condensin mutants possessing severely contracted rDNA repeats were isolated in Deltafob1 cells but not in FOB1+ cells. We also found that the condensin complex associated with the nontranscribed spacer region of rDNA with a major peak coincided with the replication fork barrier (RFB) site in a FOB1-dependent fashion. Surprisingly, condensin association with the RFB site was established during S phase and was maintained until anaphase. These results indicate that FOB1 plays a novel role in preventing repeat contraction by regulating condensin association and suggest a link between replication termination and chromosome condensation and segregation.
Abstract: With the goal of solving the whole-cell problem with Escherichia coli K-12 as a model cell, highly accurate genomes were determined for two closely related K-12 strains, MG1655 and W3110. Completion of the W3110 genome and comparison with the MG1655 genome revealed differences at 267 sites, including 251 sites with short, mostly single-nucleotide, insertions or deletions (indels) or base substitutions (totaling 358 nucleotides), in addition to 13 sites with an insertion sequence element or defective prophage in only one strain and two sites for the W3110 inversion. Direct DNA sequencing of PCR products for the 251 regions with short indel and base disparities revealed that only eight sites are true differences. The other 243 discrepancies were due to errors in the original MG1655 sequence, including 79 frameshifts, one amino-acid residue deletion, five amino-acid residue insertions, 73 missense, and 17 silent changes within coding regions. Errors in the original MG1655 sequence (<1 per 13,000 bases) were mostly within portions sequenced with out-dated technology based on radioactive chemistry.
Abstract: The goal of this group project has been to coordinate and bring up-to-date information on all genes of Escherichia coli K-12. Annotation of the genome of an organism entails identification of genes, the boundaries of genes in terms of precise start and end sites, and description of the gene products. Known and predicted functions were assigned to each gene product on the basis of experimental evidence or sequence analysis. Since both kinds of evidence are constantly expanding, no annotation is complete at any moment in time. This is a snapshot analysis based on the most recent genome sequences of two E.coli K-12 bacteria. An accurate and up-to-date description of E.coli K-12 genes is of particular importance to the scientific community because experimentally determined properties of its gene products provide fundamental information for annotation of innumerable genes of other organisms. Availability of the complete genome sequence of two K-12 strains allows comparison of their genotypes and mutant status of alleles.
Abstract: In plant meristems, each cell divides and differentiates in a spatially and temporally regulated manner, and continuous organogenesis occurs using cells derived from the meristem. We report the identification of the Arabidopsis thaliana TEBICHI (TEB) gene, which is required for regulated cell division and differentiation in meristems. The teb mutants show morphological defects, such as short roots, serrated leaves, and fasciation, as well as defective patterns of cell division and differentiation in the meristem. The TEB gene encodes a homolog of Drosophila MUS308 and mammalian DNA polymerase theta, which prevent spontaneous or DNA damage-induced production of DNA double strand breaks. As expected from the function of animal homologs, teb mutants show constitutively activated DNA damage responses. Unlike other fasciation mutants with activated DNA damage responses, however, teb mutants do not activate transcriptionally silenced genes. teb shows an accumulation of cells expressing cyclinB1;1:GUS in meristems, suggesting that constitutively activated DNA damage responses in teb lead to a defect in G2/M cell cycle progression. Furthermore, other fasciation mutants, such as fasciata2 and tonsoku/mgoun3/brushy1, also show an accumulation of cells expressing cyclinB1;1:GUS in meristems. These results suggest that cell cycle progression at G2/M is important for the regulation of the pattern of cell division and of differentiation during plant development.
Abstract: Sequences involved in the regulation of genetic and genomic processes are primarily located in noncoding regions. Identifying such cis-acting sequences from sequence data is difficult because their patterns are not readily apparent, and, to date, identification has concentrated on regions associated with gene-coding functions. Here, we used phylogenetic footprinting to look for gene-independent noncoding elements in the ribosomal RNA gene repeats from several Saccharomyces species. Similarity plots of ribosomal intergenic spacer alignments from six closely related Saccharomyces species allowed the identification of previously characterized functional elements, such as the origin-of-replication and replication-fork barrier sites, demonstrating that this method is a powerful predictor of noncoding functional elements. Seventeen previously uncharacterized elements, showing high levels of conservation, were also discovered. The conservation of these elements suggests that they are functional, and we demonstrate the functionality of two classes of these elements, a putative bidirectional noncoding promoter and a series of conserved peaks with matches to the origin-of-replication core consensus. Our results paint a comprehensive picture of the functionality of the Saccharomyces ribosomal intergenic region and demonstrate that functional elements not involved in gene-coding function can be identified by using comparative genomics based on sequence conservation.
Abstract: Gene amplification is involved in various biological phenomena such as cancer development and drug resistance. However, the mechanism is largely unknown because of the complexity of the amplification process. We describe a gene amplification system in Saccharomyces cerevisiae that is based on double rolling-circle replication utilizing break-induced replication. This system produced three types of amplification products. Type-1 products contain 5-7 inverted copies of the amplification marker, leu2d. Type-2 products contain 13 to approximately 100 copies of leu2d (up to approximately 730 kb increase) with a novel arrangement present as randomly oriented sequences flanked by inverted leu2d copies. Type-3 products are acentric multicopy minichromosomes carrying leu2d. Structures of type-2 and -3 products resemble those of homogeneously staining region and double minutes of higher eukaryotes, respectively. Interestingly, products analogous to these were generated at low frequency without deliberate DNA cleavage. These features strongly suggest that the processes described here may contribute to natural gene amplification in higher eukaryotes.
Abstract: It is known that mutations in gene SIR2 increase and those in FOB1 decrease recombination within rDNA repeats as assayed by marker loss or extrachromosomal rDNA circle formation. SIR2-dependent chromatin structures have been thought to inhibit access and/or function of recombination machinery in rDNA. We measured the frequency of FOB1-dependent arrest of replication forks, consequent DNA double-strand breaks, and formation of DNA molecules with Holliday junction structures, and found no significant difference between sir2Delta and SIR2 strains. Formal genetic experiments measuring mitotic recombination rates within individual rRNA genes also showed no significant difference between these two strains. Instead, we found a significant decrease in the association of cohesin subunit Mcd1p (Scc1p) to rDNA in sir2Delta relative to SIR2 strains. From these and other experiments, we conclude that SIR2 prevents unequal sister-chromatid recombination, probably by forming special cohesin structures, without significant effects on recombinational events within individual rRNA genes.
Abstract: Blockage of replication fork progression often occurs during DNA replication, and repairing and restarting stalled replication forks are essential events in all organisms for the maintenance of genome integrity. The repair system employs processing enzymes to restore the stalled fork. In Archaea Hef is a well conserved protein that specifically cleaves nicked, flapped, and fork-structured DNAs. This enzyme contains two distinct domains that are similar to the DEAH helicase family and XPF nuclease superfamily proteins. Analyses of truncated mutant proteins consisting of each domain revealed that the C-terminal nuclease domain independently recognized and incised fork-structured DNA. The N-terminal helicase domain also specifically unwound fork-structured DNA and Holliday junction DNA in the presence of ATP. Moreover, the endonuclease activity of the whole Hef protein was clearly stimulated by ATP hydrolysis catalyzed by the N-terminal domain. These enzymatic properties suggest that Hef efficiently resolves stalled replication forks by two steps, which are branch point transfer to the 5'-end of the nascent lagging strand by the N-terminal helicase followed by template strand incision for leading strand synthesis by the C-terminal endonuclease.
Abstract: Using an in vivo plasmid transformation method, we have determined the DNA sequences recognized by the KpnAI, StySEAI, StySENI and StySGI R-M systems from Klebsiella oxytoca strain M5a1, Salmonella eastbourne, Salmonella enteritidis and Salmonella gelsenkirchen, respectively. These type I restriction-modification systems were originally identified using traditional phage assay, and described here is the plasmid transformation test and computer program used to determine their DNA recognition sequences. For this test, we constructed two sets of plasmids, pL and pE, that contain phage lambda and Escherichia coli K-12 chromosomal DNA fragments, respectively. Further, using the methylation sensitivities of various known type II restriction enzymes, we identified the target adenines for methylation (listed in bold italics below as A or T in case of the complementary strand). The recognition sequence and methylation sites are GAA(6N)TGCC (KpnAI), ACA(6N)TYCA (StySEAI), CGA(6N)TACC (StySENI) and TAAC(7N)RTCG (StySGI). These DNA recognition sequences all have a typical type I bipartite pattern and represent three novel specificities and one isoschizomer (StySENI). For confirmation, oligonucleotides containing each of the predicted sequences were synthesized, cloned into plasmid pMECA and transformed into each strain, resulting in a large reduction in efficiency of transformation (EOT).
Abstract: Recombination hotspots are DNA sequences which enhance recombination around that region. HOT1 is one of the best-studied mitotic hotspots in yeast. HOT1 includes a RNA polymerase I (PolI) transcription promoter which is responsible for 35S ribosomal rRNA gene (rDNA) transcription. In a PolI defective mutant the HOT1 hotspot activity is abolished, therefore transcription of HOT1 is thought to be an important factor for the recombination stimulation. However, it is not clear whether the transcription itself or other pleiotropic phenotypes stimulates recombination. To investigate the role of transcription, we made a highly activated PolI transcription system in HOT1 by using a strain whose rDNA repeats are deleted (rdnDeltaDelta). In the rdnDeltaDelta strain, HOT1 transcription was increased about 14 times compared to wild-type. Recombination activity stimulated by HOT1 in this strain was also elevated, about 15 times, compared to wild-type. These results indicate that the level of PolI transcription in HOT1 determines efficiency of the recombination. Moreover, Fob1p, which is essential for both the recombination stimulation activity and transcription of HOT1, was dispensable in the rdnDeltaDelta strains. This suggests that Fob1p is functioning as a PolI transcriptional activator in the wild-type strain.
Abstract: BACKGROUND: The inhibition of DNA replication fork progression by DNA lesions can lead to cell death or genome instability. However, little is known about how such DNA lesions affect the concurrent synthesis of leading- and lagging-strand DNA catalysed by the protein machinery used in chromosomal replication. Using a system of semi-bidirectional DNA replication of an oriC plasmid that employs purified replicative enzymes and a replication-terminating protein of Escherichia coli, we examined the dynamics of the replication fork when it encounters a single abasic DNA lesion on the template DNA. RESULTS: A DNA lesion located on the lagging strand completely blocked the synthesis of the Okazaki fragment extending toward the lesion site, but did not affect the progression of the replication fork or leading-strand DNA synthesis. In contrast, a DNA lesion on the leading strand stalled the replication fork in conjunction with strongly inhibiting leading-strand synthesis. However, about two-thirds of the replication forks encountering this lesion maintained lagging-strand synthesis for about 1 kb beyond the lesion site, and the velocity with which the replication fork progressed seemed to be significantly reduced. CONCLUSIONS: The blocking DNA lesion affects DNA replication differently depending on which strand, leading or lagging, contains the lesion.
Abstract: It is speculated that the function of the replication fork barrier (RFB) site is to avoid collision between the 35S rDNA transcription machinery and the DNA replication fork, because the RFB site is located near the 3'-end of the gene and inhibits progression of the replication fork moving in the opposite direction to the transcription machinery. However, the collision has never been observed in a blockless (fob1) mutant with 150 copies of rDNA. The gene FOB1 was shown previously to be required for replication fork blocking activity at the RFB site, and also for the rDNA copy number variation through unequal sister-chromatid recombination. This study documents the detection of fork collision in an fob1 derivative with reduced rDNA copy number (approximately 20) using two-dimensional agarose gel electrophoresis. This suggests that most of these reduced copies are actively transcribed. The collision was dependent on the transcription by RNA polymerase I. In addition, the transcription stimulated rDNA copy number variation, and the production of the extrachromosomal rDNA circles (ERCs), whose accumulation is thought to be a cause of aging. These results suggest that such a transcription-dependent fork collision induces recombination, and may function as a general recombination trigger for multiplication of highly transcribed single-copy genes.
Abstract: In Escherichia coli, a replication fork blocking event at a DNA replication terminus (Ter) enhances homologous recombination at the nearby sister chromosomal region, converting the region into a recombination hotspot, Hot, site. Using a RNaseH negative (rnhA-) mutant, we identified eight kinds of Hot DNAs (HotA-H). Among these, enhanced recombination of three kinds of Hot DNAs (HotA-C) was dependent on fork blocking events at Ter sites. In the present study, we examined whether HotA DNAs are amplified when circular DNA (HotA plus a drug-resistance DNA) is inserted into the homologous region on the chromosome of a rnhA- mutant. The resulting HotA DNA transformants were analysed using pulsed-field gel electrophoresis, fluorescence in situ hybridization and DNA microarray technique. The following results were obtained: (i) HotA DNA is amplified by about 40-fold on average; (ii) whereas 90% of the cells contain about 6-10 copies of HotA DNA, the remaining 10% of cells have as many as several hundred HotA copies; and (iii) amplification is detected in all other Hot DNAs, among which HotB and HotG DNAs are amplified to the same level as HotA. Furthermore, HotL DNA, which is activated by blocking the clockwise oriC-starting replication fork at the artificially inserted TerL site in the fork-blocked strain with a rnhA+ background, is also amplified, but is not amplified in the non-blocked strain. From these data, we propose a model that can explain production of three distinct forms of Hot DNA molecules by the following three recombination pathways: (i) unequal intersister recombination; (ii) intrasister recombination, followed by rolling-circle replication; and (iii) intrasister recombination, producing circular DNA molecules.
Abstract: BACKGROUND: The analysis of homologous recombination in the tandemly repeating rDNA array of Saccharomyces cerevisiae should provide useful information about the stability of not only the rDNA repeat but also the abundant repeated sequences on higher eukaryotic genomes. However, the data obtained so far are not yet conclusive, due to the absence of a reliable assay for detecting products of recombination in the rDNA array. RESULTS: We developed an assay method to detect the products of unequal sister-chromatid recombination (marker-duplication products) in yeast rDNA. This assay, together with the circular rDNA detection assay, was used for the analysis. Marker-duplication occurred throughout the rDNA cluster, preferentially between nearby repeat units. The FOB1 and RAD52 genes were required for both types of recombinant formation. FOB1 showed a gene dosage effect on not only the amounts of both recombinants, but also on the copy number of the repeat. However, unlike the RAD52 gene, the FOB1 gene was not involved in homologous recombination in a non-rDNA locus. In addition, the marker-duplication products were drastically decreased in the mre11 mutant. CONCLUSION: Our data demonstrate that FOB1- and RAD52-dependent homologous recombination cause the gain and loss of a few copies of the rDNA unit, and this must be a basic mechanism responsible for amplification and reduction of the rDNA copy number. In addition, FOB1 may also play a role in the copy number regulation of rDNA tandem repeats.
Abstract: Saccharomyces cerevisiae carries approximately 150 ribosomal DNA (rDNA) copies in tandem repeats. Each repeat consists of the 35S rRNA gene, the NTS1 spacer, the 5S rRNA gene, and the NTS2 spacer. The FOB1 gene was previously shown to be required for replication fork block (RFB) activity at the RFB site in NTS1, for recombination hot spot (HOT1) activity, and for rDNA repeat expansion and contraction. We have constructed a strain in which the majority of rDNA repeats are deleted, leaving two copies of rDNA covering the 5S-NTS2-35S region and a single intact NTS1, and whose growth is supported by a helper plasmid carrying, in addition to the 5S rRNA gene, the 35S rRNA coding region fused to the GAL7 promoter. This strain carries a fob1 mutation, and an extensive expansion of chromosomal rDNA repeats was demonstrated by introducing the missing FOB1 gene by transformation. Mutational analysis using this system showed that not only the RFB site but also the adjacent approximately 400-bp region in NTS1 (together called the EXP region) are required for the FOB1-dependent repeat expansion. This approximately 400-bp DNA element is not required for the RFB activity or the HOT1 activity and therefore defines a function unique to rDNA repeat expansion (and presumably contraction) separate from HOT1 and RFB activities.
Abstract: At the end of the 35S rRNA gene within ribosomal DNA (rDNA) repeats in Saccharomyces cerevisiae lies an enhancer that has been shown to greatly stimulate rDNA transcription in ectopic reporter systems. We found, however, that the enhancer is not necessary for normal levels of rRNA synthesis from chromosomal rDNA or for cell growth. Yeast strains which have the entire enhancer from rDNA deleted did not show any defects in growth or rRNA synthesis. We found that the stimulatory activity of the enhancer for ectopic reporters is not observed in cells with disrupted nucleolar structures, suggesting that reporter genes are in general poorly accessible to RNA polymerase I (Pol I) machinery in the nucleolus and that the enhancer improves accessibility. We also found that a fob1 mutation abolishes transcription from the enhancer-dependent rDNA promoter integrated at the HIS4 locus without any effect on transcription from chromosomal rDNA. FOB1 is required for recombination hot spot (HOT1) activity, which also requires the enhancer region, and for recombination within rDNA repeats. We suggest that Fob1 protein stimulates interactions between rDNA repeats through the enhancer region, thus helping ectopic rDNA promoters to recruit the Pol I machinery normally present in the nucleolus.
Abstract: The problem of the low frequency of homologous recombination observed in higher plants has been approached in several ways. Here, we report a new strategy to enhance homologous recombination in Arabidopsis. In Escherichia coli and Saccharomyces cerevisiae, hotspots that enhance homologous recombination nearby have been identified in regions close to sites associated with the blocking of DNA replication forks or with intensive transcriptional activity. In yeast, a recombination hotspot (HOT1) was found in a region spanning two non-transcribed spacers (NTS) between ribosomal RNA genes (rDNA), which contains both a replication fork barrier ( RFB) and the promoter for transcription of the 35S rRNA gene. Since rDNA has a common structure among eukaryotes, we analyzed the effect of the endogenous NTS on homologous mitotic recombination in a higher plant. We constructed transgenic lines of Arabidopsis containing this NTS and a recombination substrate, in which two 3'- and 5'-deleted uidA (beta-glucuronidase) genes with partially overlapping sequences are separated by a Hyg(r) gene. Reconstitution of functional uidA genes by homologous recombination was monitored by histochemical GUS staining. We found that recombination occurred more frequently in all organs tested in F (Fork block) lines transgenic for the NTS than in C (Control) lines without the NTS. The average number of GUS+ spots on leaves in F lines was more than nine-fold higher than in C lines. Furthermore, by genomic Southern analysis, post-recombinational molecules were detected in a transgenic line, F43, which had an extremely high number of GUS+ blue spots. These results strongly suggest that NTS-dependent enhancement of homologous recombination may be a common feature of higher plants as well as yeast.
Abstract: Completion of the genome sequence of the model bacterium Escherichia coli has produced nearly 2000 open reading frames (ORFs) that remain to be functionally characterized. To accomplish this goal, we have organized a working project team in Japan and have begun construction of clones containing each of the putative ORFs. The procedure has been conceived such that we shall be able to perform systematic analysis of the shut-off as well as forced expression in vivo of each ORF and purification of its protein product for further biochemical studies. In addition, we have started a collection of various genetic and biochemical data of E. coli published in the past, and analyses of the data from a bio-informatics point of view. Thus, we aim at reaching complete understanding of this model organism in the near future.
Abstract: A new model system for the study of the SOS response has been developed. In this system the response is induced by blocking the replication fork at a Ter site located in pUC-derived plasmids. Blockage of the fork is dependent on the expression of the Ter binding protein, Tus, encoded on another plasmid, in which the tus gene is under the control of the ara promoter. SOS induction can, therefore, be controlled by arabinose. The extent of the SOS response was monitored by measuring the activity of beta-galactosidase, expressed from a lacZ gene fused to the 5' region of the sfiA gene, a typical SOS-responsive gene. Expression of the fusion gene is completely dependent on recA+ and lexA+ genes. Using this system, we found that the distance between the ori and Ter sites is directly correlated with the strength of SOS induction. The properties of this system are discussed.
Abstract: Saccharomyces cerevisiae carries approximately 150 copies of rDNA in tandem repeats. It was found that the absence of an essential subunit of RNA polymerase I (Pol I) in rpa135 deletion mutants triggers a gradual decrease in rDNA repeat number to about one-half the normal level. Reintroduction of the missing RPA135 gene induced a gradual increase in repeat number back to the normal level. Gene FOB1 was shown to be essential for both the decrease and increase of rDNA repeats. FOB1 was shown previously to be required for replication fork blocking (RFB) activity at RFB site in rDNA and for recombination hot-spot (HOT1) activity. Thus, DNA replication fork blockage appears to stimulate recombination and play an essential role in rDNA expansion/contraction and sequence homogenization, and possibly, in the instability of repeated sequences in general. RNA Pol I, on the other hand, appears to control repeat numbers, perhaps by stabilizing rDNA with the normal repeat numbers as a stable nucleolar structure.
Abstract: The contiguous 874.423 base pair sequence corresponding to the 50.0-68.8 min region on the genetic map of the Escherichia coli K-12 (W3110) was constructed by the determination of DNA sequences in the 50.0-57.9 min region (360 kb) and two large (100 kb in all) and five short gaps in the 57.9-68.8 min region whose sequences had been registered in the DNA databases. We analyzed its sequence features and found that this region contained at least 894 potential open reading frames (ORFs), of which 346 (38.7%) were previously reported, 158 (17.7%) were homologous to other known genes, 232 (26.0%) were identical or similar to hypothetical genes registered in databases, and the remaining 158 (17.7%) showed no significant similarity to any other genes. A homology search of the ORFs also identified several new gene clusters. Those include two clusters of fimbrial genes, a gene cluster of three genes encoding homologues of the human long chain fatty acid degradation enzyme complex in the mitochondrial membrane, a cluster of at least nine genes involved in the utilization of ethanolamine, a cluster of the secondary set of 11 hyc genes participating in the formate hydrogenlyase reaction and a cluster of five genes coding for the homologues of degradation enzymes for aromatic hydrocarbons in Pseudomonas putida. We also noted a variety of novel genes, including two ORFs, which were homologous to the putative genes encoding xanthine dehydrogenase in the fly and a protein responsible for axonal guidance and outgrowth of the rat, mouse and nematode. An isoleucine tRNA gene, designated ileY, was also newly identified at 60.0 min.
Abstract: BACKGROUND: In the rRNA gene cluster on the Saccharomyces cerevisiae chromosome XII, a unique site that blocks progression of the replication fork exists in a single unit of the repeats. The site RFB (replication fork blocking) is located within one of two cis-elements present in a nontranscribed region of each repeated unit, which are required for obtaining maximal activity of a hotspot (HOT1) dependent recombination. RESULTS: To investigate the correlation between replication fork blocking at RFB and homologous recombination at HOT1, we have isolated Hot1-defective mutants and examined their ability for fork blocking at RFB. Amongst 23 isolated mutants, four were found to be defective in both abilities. Genetic analysis of the mutants reveals that a single mutation, named fob1 (fork blocking less), is responsible for the defects in both abilities. The FOB1 gene is located on chromosome IV and has no homology with any other genes listed in DNA data banks. CONCLUSION: The pleiotropic effect of the fob1 mutation suggests that homologous recombination at HOT1 is closely linked with DNA replication fork blocking event at RFB.
Abstract: The 718,122 base pair sequence of the Escherichia coli K-12 genome corresponding to the region from 12.7 to 28.0 minutes on the genetic map is described. This region contains at least 681 potential open reading frames, of which 277 (41%) have been previously identified, 147 (22%) are homologous to other known genes, 139 (20%) are identical or similar to the hypothetical genes registered in databases, and the remaining 118 (17%) do not show a significant similarity to any other gene. In this region, we assigned a cluster of cit genes encoding multienzyme citrate lyase, two clusters of fimbrial genes and a set of lysogenic phage genes encoding integrase, excisionase and repressor in the e14 genetic element. In addition, a new valine tRNA gene, designated valZ, and a family of long directly repeated sequences, LDR-A, -B and -C, were found.
Abstract: The 465,813 base pair sequence corresponding to the 40.1-50.0 min region on the genetic map of Escherichia coli K-12 (W3110) was determined. Analysis of the sequence revealed that this region contained at least 466 potential open reading frames, of which 187 (40%) were previously reported, 105 (23%) were homologous to other known genes, 103 (22%) were identical or similar to hypothetical genes registered in databases, and the remaining 71 (15%) did not show a significant similarity to any other gene. At the 45.2-46.0 min region, we found a very large cluster of about 30 genes, whose functions are involved in the biosynthesis of polysaccharides as the components of outer membranes. In addition, we identified a new asn-tRNA gene, designated asnW, between the asnT and asnU genes and a new lysogenic phage attachment site as the cis-element.
Abstract: The 569,750 base pair sequence corresponding to the 28.0-40.1 min region on the genetic map of Escherichia coli K-12 (W3110) was determined. This region includes the replication terminus region and contained at least 549 potential open reading frames. Among them, 160 (29%) were previously reported, 174 (32%) were homologous to other known genes, 102 (18%) were identical or similar to hypothetical genes registered in databases, and the remaining 113 (21%) did not show a significant similarity to any other gene. Of interest was the finding of a large number of genes and gene clusters in and near the replication termination region which had been thought to be genetically silent. Those included a cluster of genes for fatty acid beta-oxidation, the third copy of the pot (spermidine/putrescine transport system) gene cluster, the second dpp (dipeptide transport system) operon, the second dsm (anaerobic dimethyl sulfoxide reductase) operon, a cluster of fim (fimbrial) genes and a DNA helicase-like gene with a high molecular weight. In addition, we found the dnaC- and dnaT-like genes in the cryptic prophage, Rac, and a number of genes originated probably from plasmids.
Abstract: In E. coli rnh- mutants we identified chromosome-derived, specific DNA fragments termed Hot DNA. When the DNA in the ccc form is integrated into the E. coli genome by homologous recombination to form a directly repeated structure, a striking enhancement of excisional recombination between the repeats occurs. We obtained 8 groups of such Hot DNA, 7 of which were clustered in a narrow region called the replication terminus region (about 280 kb) on the circular E. coli genome. A Ter site can impede the replication fork in a polar fashion. The six Ter sites are approximately symmetrical in the terminus and surrounding region. To block the fork at the Ter site, a protein factor, Ter binding protein encoded in the tau (or tus) gene, is required. In tau- cells, Hot activity of HotA, B, and C DNAs disappears, thereby indicating that the Hot activity is fork arrest-dependent. Other Hot activities were tau-independent. In addition, for at least HotA activity, the presence of Chi, and E. coli recombinational hotspot sequence, is required; the Chi dependent HotA activity was detected in a wild type strain but to a lesser extent than that in the rnh- mutant. To explain the HotA phenomenon at the molecular level, we propose a model in which a ds-break occurs at the replication fork arrested at the Ter site. Our recent data that HOT1, a yeast recombinational hotspot, may also depend on the fork blocking event for activity, suggests that a similar ds-break occurs in both eucaryotes and procaryotes.
Abstract: To examine the physiological effects of DNA replication arrest at the terminus (Ter), we constructed a replication-blocked Escherichia coli strain so that both bidirectional replication forks would be impeded at two flanking Ter sites, one artificial and the other natural. While the blocked strain grew slightly more slowly than a control strain, it had abnormal phenotypes similar to those of E. coli dam mutants, i.e., hyper-Rec phenotype, recA(+)- and recB+ (C+)-dependent growth, and constitutive SOS induction. The observation that these two apparently unrelated mutants cause similar phenotypes led us to design a model. We propose that the following sequential events may occur in both strains. A double-strand (ds) break occurs at the blocked replication fork in the blocked strain and at the ongoing fork in the dam mutant, through which RecBCD enzyme enters and degrades the ds DNA molecule, and the degradation product serves as the signal molecule for SOS induction. When RecBCD enzyme meets an appropriately oriented Chi sequence, its DNase activity is converted to recombinase enzyme, which is able to repair the ds end, recombinationally. this model (i) explains the puzzling phenotype of recA and recB (C) mutants and the SOS-inducing phenotype of polA, lig, and dna mutants under restrictive conditions, (ii) provides an interpretation for the role of the Chi sequence, and (iii) suggests a possible key role for homologous recombination with regard to cell survival following the arrest of DNA replication.
Abstract: In Escherichia coli, eight kinds of chromosome-derived DNA fragments (named Hot DNA) were found to exhibit homologous recombinational hotspot activity, with the following properties. (i) The Hot activities of all Hot DNAs were enhanced extensively under RNase H-defective (rnh) conditions. (ii) Seven Hot DNAs were clustered at the DNA replication terminus region on the E. coli chromosome and had Chi activities (H. Nishitani, M. Hidaka, and T. Horiuchi, Mol. Gen. Genet. 240:307-314, 1993). Hot activities of HotA, -B, and -C, the locations of which were close to three DNA replication terminus sites, the TerB, -A, and -C sites, respectively, disappeared when terminus-binding (Tau or Tus) protein was defective, thereby suggesting that their Hot activities are termination event dependent. Other Hot groups showed termination-independent Hot activities. In addition, at least HotA activity proved to be dependent on a Chi sequence, because mutational destruction of the Chi sequence on the HotA DNA fragment resulted in disappearance of the HotA activity. The HotA activity which had disappeared was reactivated by insertion of a new, properly oriented Chi sequence at the position between the HotA DNA and the TerB site. On the basis of these observations and positional and orientational relationships between the Chi and the Ter sequences, we propose a model in which the DNA replication fork blocked at the Ter site provides an entrance for the RecBCD enzyme into duplex DNA.
Abstract: To clone new replication origin(s) activated under RNase H-defective (rnh-) conditions in Escherichia coli cells, whole chromosomal DNA digested with EcoRI was to with a Kmr DNA fragment and transformed into an rnh- derivative host. From the Kmr transformants, we obtained eight kinds of plasmid-like DNA, each of which contained a specific DNA fragment, termed "Hot", derived from the E. coli genome. Seven of the Hot DNAs (HotA-G) mapped to various sites within a narrow DNA replication termination region (about 280 kb), without any particular selection. Because Hot DNA could not be transformed into a mutant strain in which the corresponding Hot region had been deleted from the chromosome, the Hot DNA, though obtained as covalently closed circular (ccc) DNA, must have arisen by excision from the host chromosome into which it had initially integrated, rather than by autonomous replication of the transformed species. While Hot DNA does not have a weak replication origin it does have a strong recombinational hotspot active in the absence of RNase H. This notion is supported by the finding that Chi activity was present on all Hot DNAs tested and no Hot-positive clone without Chi activity was obtained, with the exception of a DNA clone carrying the dif site.
Abstract: DNA replication terminus (ter)-binding protein (TBP) in Escherichia coli binds specifically to the terminus (ter) site, and the resulting complex severely blocks DNA replication in an unique orientation by inhibiting the action of helicases. To generalize the intrinsic nature of the orientated ter-TBP complex against various helicases, we tested the potential of the complex to inhibit the action of three helicases, DNA helicase I, simian virus 40 (SV40) large tumor (T) antigen, and helicase B, derived from F plasmid, SV40, and mouse FM3A cell, respectively. The complex impeded the unwinding activities of all tested helicases in a specific orientation, with the same polarity observed in case of blockage of a replication fork, and, as a result, there was a block of SV40 DNA replication in both crude and purified enzyme systems in vitro. As the specificity in polarity of inhibition extends to heterologous systems, there may be common structure/mechanism features in helicases.
Abstract: The yeast genome has DNA replication fork blocking sites, that we have named sog sites, in the ribosomal RNA gene (rDNA) cluster. These are located at the 3' end of the 35S rRNA transcription unit and they block replication fork movement in a direction opposite to that of RNA polymerase I. We cloned this replication blocking site into a YEp-type plasmid and analyzed DNA replication intermediates, using two-dimensional (2D) agarose gel electrophoresis. The blocking activity remained even on a plasmid not involved in 35S rRNA transcription and inhibited fork movement in the same polar fashion as on the yeast chromosome. To define the site further, smaller fragments were subcloned into the YEp-type plasmid. A small 109 bp region exhibited sog activity and was located near the enhancer region for 35S rRNA transcription. It overlaps an essential element of the recombinational hot spot HOT1.
Abstract: To search for heretofore unidentified DNA replication termination (Ter) sites on the Escherichia coli chromosome, we screened the entire Kohara lambda bacteriophage library using as probes the four known 22-bp Ter sequences. We found a Ter site, which we named TerE, located at 23.2 min on the linkage map. TerE inhibits only counterclockwise DNA replication. Macroscopically, five Ter sites are located in a periodic arrangement on the genome.
Abstract: DNA polymerase III holoenzyme is responsible for chromosomal DNA synthesis in Escherichia coli and seems to be a major determinant of the fidelity of replication of this organism. Among ten different subunits of the holoenzyme, the alpha subunit, encoded by the dnaE gene, has a polymerase activity, while the epsilon subunit, encoded by the dnaQ gene, is a proofreader with a 3'-5' exonuclease activity. Using poly(dA)/oligo(dT)20 as a template-primer, misincorporation of dGMP, dCMP, and dAMP by the alpha subunit and exonucleolytic editing of those mispairs by the epsilon subunit were investigated. When the polymerization reaction was performed with the alpha subunit, dCMP and dGMP but not dAMP were misincorporated. This would suggest that the polymerase might have a base-selecting function to avoid dA:dA mispairing. A subassembly of the DNA polymerase III consisting of alpha, epsilon, and theta subunits misincorporated only dGMP. This would imply that the proofreading function of the epsilon subunit may correct the dC:dA but not the dG:dA mispair. Addition of a protein encoded by the mutT gene, defects of which cause AT to CG transversions in vivo, diminished the misincorporation of dGMP onto poly(dA) template by the alpha subunit. A dGTPase activity was associated with the MutT protein. The significance of the dGTPase activity in the prevention of dG:dA mispairing is discussed.
Abstract: Activity binding specifically to the 22 bp of the DNA replication terminus (ter) sequence on plasmid R6K and the Escherichia coli genome was detected in the crude extract of E. coli cells. This activity was inactivated by heat or by protease but not by RNase treatments. Overproduction of the ter binding activity was observed when the extract was prepared from the cell carrying a plasmid with a chromosomal-derived 5.0 kb EcoRI fragment, on which one of the four terC sites, terC2, was also located. By mutagenesis of the 5.0 kb fragment on the plasmid with transposon Tn3 and subsequent replacement of the corresponding chromosomal region with the resulting mutant alleles, we isolated tau- mutants completely defective in ter binding activity. These mutants simultaneously lost the activity to block the progress of the DNA replication fork at any ter site, on the genome or the plasmid. It would thus appear that the ter binding protein plays an essential role in the termination reaction, at the ter sites.
Abstract: DNA polymerase III has been recognized as the required replication enzyme in Escherichia coli. The synthesis subunit of DNA polymerase III holoenzyme (alpha subunit) is encoded by the dnaE gene. We have reported that E. coli cells can survive and grow in the absence of a functional dnaE gene product if DNA polymerase I and the pcbA1 mutation are present. Existing mutations in the dnaE gene have been conditionally defective thermolabile mutations. We report here construction of nonsense mutations in the dnaE gene by use of a temperature-sensitive suppressor mutation to permit survival at the permissive temperature (32 degrees C). Introduction of the pcbA1 mutation eliminated the temperature-sensitive phenotype. We confirmed by immunoblotting the lack of detectable alpha subunit at 43 degrees C.
Abstract: Occurrence of the transversion mutation A.T to C.G is specifically enhanced in Escherichia coli mutT mutants. With the aid of the cloned mutT gene, the MutT protein, which has a molecular mass of 15 kilodaltons, was overproduced and purified to near homogeneity. The protein catalyzes hydrolysis of dGTP to dGMP. dGDP and GTP were also hydrolyzed by the protein, but at a lower rate than seen with dGTP. No other deoxynucleoside triphosphates were hydrolyzed. Using poly(dA).(dT)20 as a template-primer, we investigated the misincorporation of dGMP, dCMP, and dAMP by the alpha subunit and the core of E. coli DNA polymerase III. When the polymerization reaction was performed with the alpha subunit, both dCMP and dGMP were misincorporated. The core, composed of alpha, epsilon, and theta subunits, misincorporated only dGMP. This would imply that the proofreading function of the epsilon subunit of DNA polymerase III may correct the dC.dA mispair but not the dG.dA mispair. Misincorporation of dAMP was not observed in reactions with the alpha subunit or core. The misincorporation of dGMP, but not dCMP, was almost completely suppressed by adding purified MutT protein to the reaction mixture. Under these conditions, only a portion of dGTP present in the reaction mixture was degraded. It is therefore likely that the MutT protein may prevent dGMP misincorporation by degrading a specific form of dGTP, probably the syn form, which can pair with deoxyadenosine.
Abstract: Identification of the consensus sequence for termination of replication (ter) in Escherichia coli and the isolation of the ter-binding protein (TBP) allowed us to test their effects on replication forks initiated at the unique origin of the E. coli chromosome (oriC) in a purified enzyme system. Replication was severely impeded by ter in a unique orientation when purified TBP was supplied to bind it. The target for blockage within the replication complex can now be ascribed to the inability of dnaB helicase to separate the duplex strands when it encounters ter bound by TBP. Other helicases, such as rep and uvrD proteins, that translocate on DNA and displace strands in the direction opposite to that of dnaB protein are also blocked, but only when the TBP-bound ter is oriented in the other direction. From these results, we infer that the orientation of ter confers a particular polarity on the TBP seated on it, such that a helicase is blocked when it confronts TBP from one side, but can act, presumably by displacing TBP, when facing its other side. Thus, the intrinsic nature of the oriented TBP-ter complex is responsible for impeding the helicases, rather than any protein-protein interactions.
Abstract: In Escherichia coli cells, there is a protein that specifically binds to DNA replication terminus (ter) sites on the host and plasmid genome and then blocks progress of the DNA replication fork. We reported that extract of the cells carrying the plasmid with the tau gene, which was identified to be an essential gene for the termination reaction at the ter site, contained about an 8-fold increase in ter-binding activity of the plasmid-free cells. With improvement of the promoter region of the tau gene on the plasmid by site-directed mutagenesis, the host cells produced the ter-binding protein (Ter protein) over 2,000-fold. Using these over-producing cells as the enzyme source, the Ter protein was purified to apparent homogeneity. Molecular mass 36,000, amino-terminal amino acid sequence (45 residues) and composition of the protein were in good agreement with those deduced from DNA sequence of the tau gene. Footprinting using the purified Ter protein revealed a specific binding to the ter sequences.
Abstract: The DNA replication terminus (terR) of the R6K plasmid located on a 216 bp Alul fragment (Alu216) can block progress of the DNA replication fork. We previously developed an electrophoresis assay that allows detection of terminus activity on any DNA fragment cloned in the pUC vector. For precise identification of terR, we tested Alu216, its subfragments, and synthetic oligonucleotides by this assay. We found terR to be composed of a pair of separable sites, terR1 and terR2, each of which can block the DNA replication fork traveling in a specific but not the opposite direction. Both terR sites were composed of 22 nucleotides containing the repeated 20 bp sequence 5'-TAGTTACAACAC(A or T) CAA(G or T) AGA-3', located 73 bp apart in the inverted position of Alu216. A DNA homology search suggested that the R6K plasmid and the E. coli chromosome share a common termination system.
Abstract: Using the "Ter assay" we developed, three separate replication terminus (terC1, terC2, and terC3) sites on the E. coli chromosome were identified. The locations are at 28.3, 35.6, and 33.9 min on the linkage map, respectively. The terC1 site can block the counter-clockwise replication fork only, while the terC2 and terC3 sites inhibit the clockwise fork traveling on the chromosome. DNA sequences of the terC sites required for termination of DNA replication are highly homologous to those of terminus (terR) sites of the R6K plasmid, and the 21 bp consensus DNA sequence of terC is 5'-(A or T) TTAGTTACAACAT (A or C) CT (A or T) (A or T) (A or T) T-3'. In addition, all Ter active pUC-terC plasmids had a low copy number and were unstable in the host cells.
Abstract: A 4.32 kb DNA fragment, on which the DNA replication terminus (terR) site of plasmid R 6K was located, was inserted into the unique EcoRI site of plasmid pUC9. To detect replication intermediate molecules with a replication fork halted at the terR site, a cell DNA extract was digested with EcoRI, electrophoresed through an agarose gel and stained with ethidium bromide. In addition to two major bands, one derived from vector DNA and the other from the ter insert fragment, two extra minor bands were detected. Following DNA-DNA hybridization and electron microscopic observation we concluded that the two minor bands corresponded to the two Y-shaped molecules, produced from the theta-shaped intermediate molecules by EcoRI digestion.
Abstract: The Escherichia coli mutator gene mutT, which causes A:T----C:G transversion, was cloned in pBR 322. mutT+ plasmids carry a 0.9 kb PvuII DNA fragment derived from the E. coli chromosome. Specific labelling of plasmid-encoded proteins by the maxicell method revealed that mutT codes for a polypeptide of about 15,000 daltons. The protein was overproduced when the mutT gene was placed under the control of the lac regulatory region on a multicopy runaway plasmid. The nucleotide sequence of the mutT gene was determined by the dideoxy method.
Abstract: We report a new tRNA(1Asp) gene near the dnaQ gene, which is located at 5 min on the Escherichia coli linkage map. We named it aspV. The sequence corresponding to the mature tRNA is identical with that of the two previously identified tRNA(1Asp) genes (aspT and aspU), but there is no homology in the sequences of their 3'- and 5'-flanking regions.
Abstract: The nucleotide sequences of the recessive dnaQ49 and the dominant mutD5 mutator were determined. The dnaQ49 mutator has a single base substitution in the dnaQ gene, thus causing one amino acid change, 96Val (GTG)----Gly (GGG), in the DnaQ protein (epsilon subunit of DNA polymerase III holoenzyme). The mutD5 mutator possesses two base substitutions in the same gene, resulting in two amino acid changes, 73Leu (TTG)----Trp (TGG) and 164Ala (GCA)----Val (GTA), which were designated the mutD52 and mutD51 mutations, respectively. Construction of chimaeric genes carrying one or two of these mutations revealed: either mutD51 or mutD52 alone causes the dominant mutator phenotype when present in a multi-copy plasmid; mutator phenotype when present in a low-copy plasmid; the dominant mutD51 mutator activity is suppressed by the dnaQ49 mutation when both mutations are present in the same gene. Based on these findings, we devised a model for the action of these mutators.
Abstract: The Escherichia coli dnaE gene, which encodes the alpha subunit of DNA polymerase III (pol III) holoenzyme, has been cloned in a plasmid containing the PL promoter of phage lambda and thermally induced to overproduce the alpha subunit. In cells carrying this plasmid (pKH167), the alpha subunit was amplified, after heat induction, to a level of about 0.2% of the total cellular protein. Polymerase activity was assayed in three ways: (i) gap-filling by pol III holoenzyme and subassemblies of it, (ii) the extensive replication of a primed, single-stranded DNA circle only by pol III holoenzyme, and (iii) complementation of a crude, inactive pol III holoenzyme (temperature-sensitive dnaE mutant fraction) in replication of a primed, single-stranded DNA circle. Amplification of the alpha subunit raised the polymerase level 10-fold in assay (i), indicative of the dependence of pol III gap-filling activity on this polypeptide; pol III holoenzyme activity remained unaffected (assay (ii)), but the complementation activity was raised 5-fold (assay (iii)). Thus, the elevated alpha subunit (free or in a subassembly form) can substitute in vitro for a defective alpha subunit in pol III holoenzyme, but cannot increase the in vivo level of about eight pol III holoenzyme molecules per cell. This low level of pol III holoenzyme is fixed in wild type cells (bearing no plasmid) despite the presence of a 5-fold excess of the alpha subunit, as inferred from the various assays. These results suggest that the low level of pol III holoenzyme is determined by a factor or factors other than the level of the alpha subunit.
Abstract: Mutants of Escherichia coli completely deficient in RNase H activity were isolated by inserting transposon Tn3 into the structural gene for RNase H, rnh, and its promoter. These rnh- mutants exhibited the following phenotypes; (1) the mutants grew fairly normally, (2) rnh- cells could be transformed with ColE1 derivative plasmids, pBR322 and pML21, though the plasmids were relatively unstable, under non selective conditions, (3) rnh- mutations partially suppressed the temperature-sensitive phenotype of plasmid pSC301, a DNA replication initiation mutant derived from pSC101, (4) rnh- mutations suppressed the temperature-sensitive growth character of dnaAts mutant, (5) rnh- cells showed continued DNA synthesis in the presence of chloramphenicol (stable DNA replication). Based on these findings we propose a model for a role of RNase H in the initiation of chromosomal DNA replication. We suggest that two types of RNA primers for initiation of DNA replication are synthesized in a dnaA/oriC-dependent and -independent manner and that only the dnaA/oriC-dependent primer is involved in the normal DNA replication since the dnaA/oriC independent primer is selectively degraded by RNase H.
Abstract: By using localized mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, we isolated 39 temperature-sensitive growth mutants that exhibited high mutability when the bacteria were grown at the permissive temperature. Two of the mutations, dnaQ186 and dnaQ231, were shown to be new alleles of the dnaQ gene by genetic mapping and complementation tests with the dnaQ49 mutation previously isolated. They shared common properties with the dnaQ49 strain, but their mutator activity was not temperature dependent. The dnaQ mutants exhibited increased sensitivity to inhibitors of DNA gyrase and to DNA intercalating and alkylating agents.
Abstract: A 1.6-kilobase-pair DNA fragment derived from the Escherichia coli chromosome was analyzed by Tn3 transposon insertion and deletion mapping to locate a mutator gene, dnaQ (mutD), and the rnh gene that codes for RNase H. When a strong promoter, PL of lambda phage, was placed at the right- and left-side of the cloned DNA fragment, the dnaQ protein and RNase H, respectively were overproduced. These results suggested that the two genes are transcribed in opposite directions and that their promoters are located in a narrow region between the genes. Nucleotide sequence analysis confirmed this and further revealed that transcriptional and translational initiation signals for the two genes overlap. From the sequence data it was deduced that the dnaQ protein and RNase H consist of 243 and 155 triplets and have molecular weights of 27,500 and 17,500, respectively. dnaQ81 amber mutant showed two codon alterations, CAG(glutamine-195) leads to TAG(amber) and ACA(threonine-193) leads to ATA(isoleucine). The dnaQ-lacZ and the rnh-lacZ fused genes were constructed and hybrid proteins with beta-galactosidase activity were produced. From beta-galactosidase levels it was estimated that the promoter for dnaQ is 5 times more active than that for rnh.
Abstract: The two known strong mutators of Escherichia coli K12, mutD5 (Degnen & Cox, 1974) and dnaQ49 (Horiuchi et al., 1978), are located at almost the same position, at five minutes on the linkage map. To clarify the genetical and functional relationships between these two mutators, we have constructed hybrid plasmids and phages carrying dnaQ+ or mutD5 by using in vivo and in vitro recombination techniques and examined their effect on the phenotype of wild-type or mutant bacteria. The results indicated that the mutD5 mutator is dominant over the wild-type allele whereas dnaQ49 is recessive. Thus, mutD5 plasmid or mutD5 transducing lambda phage can be used to convert a wild-type strain to a highly mutable strain. Both dnaQ+ and mutD5 plasmids carried a 1.5 X 10(3) base DNA fragment derived from the E. coli chromosome and they were indistinguishable from each other by restriction enzyme analysis. Moreover, specific labeling of the plasmid-encoded proteins by the maxicell method revealed that the mutD5 plasmid codes for two proteins, one whose molecular weight is 25,000 and the other whose molecular weight is 21,000, which correspond to the dnaQ protein and RNase H, respectively. Insertion of the gamma delta sequence into the mutD gene of the plasmid resulted in disappearance of the 25,000 Mr protein. These results suggested that the dnaQ49 and mutD5 mutator are mutations that have arisen in a single gene, though they differ in many respects.
Abstract: To elucidate molecular mechanisms leading to the increment of mutation frequency by mutator genes, we have cloned the dnaQ and uvrD genes of Escherichia coli and identified their gene products. By in vitro recombination hybrid plasmids capable of complementing a dnaQ mutation were constructed. The dnaQ+ plasmids consist of a full-length pBR322 DNA and a 1.5-kilobase DNA fragment derived from the E. coli chromosome. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the 1.5-kilobase insert codes for two proteins, one with a molecular weight of 25,000 and the other with a weight of 21,000. Because insertion of the gamma delta sequence into the dnaQ gene of the plasmid resulted in disappearance of the 25,000-dalton protein, it was concluded that that protein is the dnaQ gene product. The 21,000-dalton protein was identified as RNase H. The uvrD (uvrE, recL, mutU, and pdeB) gene has been cloned with phage lambda as vector. The increased sensitivity to ultraviolet light, high mutability and conditional lethality of uvrD- strains and their derivatives were all suppressed by lysogenization of the mutant cells with lambda uvrD+. In addition to the uvrD gene, lambda uvrD+ carried the corA gene that controls transport of Mg2+, Mn2+, and Co2+ through the cell membrane. By analyzing proteins produced by the transducing phages, the uvrD and corA gene products were identified as a 75,000-dalton protein and a 37,000-dalton protein, respectively.
Abstract: By in vitro recombination we have constructed hybrid plasmids capable of complementing a conditional lethal mutator mutation, dnaQ49, in Escherichia coli K12. The dnaQ+ plasmids consist of a full-length pBR322 DNA and a 1.5-kilobase DNA fragment derived from the E. coli chromosome. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the 1.5-kilobase insert codes for two proteins, one whose molecular weight is 25,000 [the 25-kilodalton (kDal) protein] and the other whose molecular weight is 21,000 (the 21-kDal protein). Because insertion of gamma delta sequence into the dnaQ gene of the plasmid resulted in disappearance of the 25-kDal protein, it was concluded that the 25-kDal protein is the dnaQ gene product. The 21-kDal protein was identified as RNase H on the basis of the following evidence. (i) Cells harboring the dnaQ+ plasmids, with or without the gamma delta insertion in the dnaQ gene, had a 5- to 7-fold higher level of RNase H activity than cells harboring pBR322. (ii) After induction of cells that are lysogenized with dnaQ+-transducing lambda phages, RNase H activity increased considerably. A similar high level of RNase H activity was observed with transducing phages whose dnaQ function was inactivated by insertion of a transposon, Tn3, into the gene, (iii) The plasmid-encoded RNase H, labeled with [35S]methionine, was purified in a manner essentially similar to that of the chromosome-encoded enzyme. These results suggest that the dnaQ gene and the structural gene for RNase H, termed gene rnh, are closely linked and located at 5 min on the linkage map.
Abstract: The uvrD gene of Escherichia coli that controls UV sensitivity and spontaneous mutation frequency has been cloned with phage lambda as vector. The increased sensitivity to ultraviolet light (UV) of uvrD3, uvrE502, recL152, and pdeB41 mutants, high mutability of uvrD3 and pdeB41 mutants, and conditional lethality of strain TS41 that carried pdeB41, polA1, and supl26 mutations were all suppressed by lysogenization of the mutant cells with lambda uvrD+. These results were consistent with the idea that the uvrD, uvrE, recL, and pdeB mutations are alleles of the uvrD gene. In addition to the uvrD gene, lambda uvrD+ carried the corA gene that controls transport of Mg++, Mn++, and Co++ through the cell membrane. Hybrid plasmids carrying both uvrD and corA genes were also constructed by using pKY2289 as a cloning vehicle. Orientational isomers that carried the same 12.0 kb fragment in the opposite direction were equally efficient in complementing the UvrD- as well as CorA- defects of the transformed host cells, suggesting that the DNA insert contains all the genetic signals needed to express the two gene products. Insertion of the gamma delta sequence into recombinant plasmids was performed to generate appropriate restriction endonuclease target sites in the cloned DNA fragments.
Abstract: A double mutant of Escherichia coli K12 which carries a conditional lethal mutator mutation, dnaQ49 (Horiuchi et al. 1978), and a DNA polymerase III-deficient mutation, dnaE486 (Wechsler and Gross 1971), was found to be more thermolabile than was either of the dnaQ49 or dnaE486 single mutants. The double mutant is able to grow at 28 degrees C but not at 30 degrees C. Under the restrictive conditions DNA synthesis, but not protein synthesis, of the double mutant was suppressed. All the other combinations of dnaQ and dnaE mutation alleles tested so far rendered the cells thermolabile. A dnaZ mutation exerted a similar effect on the dnaQ strain. However, when non-specific temperature-sensitive growth mutations were combined with the dnaQ49 mutation, no such increase in thermosensitivity was observed. There is a possibility that the product of the dnaQ gene interacts directly with the DNA replicating enzyme complex.
Abstract: A conditional lethal mutator, dnaQ49, was found in Escherichia coli K12. The dnaQ49 mutation caused stimulation of rifampicin-, nalidixic acid- and streptomycin-resistant mutation frequencies 100 to 2000 fold at 30 degrees C and the frequencies were further increased 50 to 100 fold at 35 degrees C or higher temperatures. Cells carrying dnaQ49 were unable to grow in salt-free L-broth at 44.5 degrees C, and DNA synthesis but not protein synthesis of the cells was suppressed under the restrictive conditions. The dnaQ gene was located at about 5 min on the E. coli linkage map and the order of the genes residing in this region was determined to be ton A-dnaE-metD-dnaQ-pro A.
Abstract: Genetic analyses of an Escherichia coli K-12 mutant possessing the amber mutation lig-321 were carried out. This mutant is defective in deoxyribonucleic acid (DNA) ligase and conditionally lethal. We constructed strains harboring an F'lig+ or F'lig-321 plasmid. Genetic complementation analyses were done by using these plasmids and by constructing a lig-4/F'lig-321 merodiploid. It was shown that lig-321 does not complement lig-4, unless the former is suppressed by an amber suppressor. The same was found to be the case between lig-321 and lig-ts7. Transductional mapping of lig-321, by a four-factor cross, revealed that lig-321 is very closely linked to lig-4. The frequency of recombinants between the two alleles was not unreasonable for assuming that they arose by intragenic recombination. The lig-4 and lig-ts7 alleles are known to reside in the structural gene for DNA ligase, in which lig-321 may also be located.
