Inducible, error-free DNA repair in tsl recA mutants of E. coli

Summary Host cell reactivation and UV reactivation and mutagenesis of UV-irradiated phage were measured in tsl recA ⁺ and tsl recA host mutants. Host cell reactivation was slightly more efficient in the tsl recA strain compared to the tsl ⁺ recA strain. Phage was UV-reactivated in the tsl recA strain with about one-half the efficiency of that in the wild type strain, but there was no corresponding mutagenesis of phage. UV-reactivation was also slightly lower and mutagenesis several-fold lower than normal in the tsl recA ⁺ strain. To account for these observations, we propose that there is an inducible, error-free pathway of DNA repair in E. coli that competes with error-prone repair for repair of phage lesions.     

Nucleotide excision by E. coli DNA polymerase I in proofreading and non-proofreading modes

Escherichia coli DNA polymerase I exists in at least two distinct kinetic forms. When it binds to a template, the proofreading activity is usually switched off. As the enzyme progresses along the template, it becomes more and more competent for excision. This phenomenon introduces a link between fidelity and processivity. Processivity is best studied when the chain-length distributions of synthesized polymers are stationary. Even then, however, one cannot avoid multiple initiations on a given template by the same molecule of the enzyme. When synthesis is initiated with primers of lengths 15 or 20, a strange phenomenon is observed. It seems that the polymerase starts by hydrolyzing the primer down to a length of 7–10 nucleotides and only then starts to add nucleotides. It does so in a low-accuracy mode, suggesting that, while the exonuclease is clearly active, it does not contribute to proofreading. The warm-up of the proofreading function is therefore reinterpreted as a switch between two modes of behaviour: a mode 1 of low accuracy in which the 3′ → 5′ exonuclease, while active, is uncoupled from the polymerase and does not contribute to proofreading, and a mode 2 of high accuracy in which the exonuclease is kinetically linked to the polymerase activity.     

Replication of E. coli duplex DNA in vitro

Summary An E. coli lysate after being gently washed to remove soluble components, supports replicative DNA synthesis, if soluble proteins and the deoxyribonucleotide triphosphates are added. This DNA synthesis is dependent on ATP and on the presence of the gene products of the dnaB, dnaG, and polC (DNA polymerase III) genes. It continues at the replication forks preformed in vivo and Okazaki fragments are intermediate products of the reaction.Two different methods were used to prepare the washed DNA containing fraction. The one method involves washing of a cell lysate situated on a dialysis membrane. The other method involves DNAase treatment of a lysate and sedimentation of the degraded DNA through a glycerol gradient. Both washed preparations contain not only the DNA and the replication forks but also functional amounts of DNA polymerase III and of the dnaB gene product. Other factors, that are essential for replicative DNA synthesis, including the dnaG gene product, are washed out of the DNA containing preparations and the system is reconstituted by readdition of the soluble proteins.     

Mechanisms of spontaneous mutation in DNA repair-proficient Escherichia coli

This paper describes the DNA sequence analysis of 729 independent spontaneous lacI⁻ mutation This total is comprised of 478 novel mutations and 251 previously described events, and therefore should allow a more comprehensive view of spontaneous mutation in Escherichia coli. The spectrum is dominated by a hotspot (71% of all events). Mutations at this site consist of related addition and deletion events involving a number of repetitive sequences. Here we discuss how the frequency and proportion of these events vary in different DNA repair-deficient genetic backgrounds. The distribution of non-hotspot events includes base substitutions (38%), deletions (35%), frameshifts (14%), duplications (4%) and insertion elements (4%). G:C → A:T events dominate among base substitutions, while G:C → C:G events are the least common; the remaining types of base substitution are equally represented. Among deletions, a significant number do not display repeated sequences at their endpoints (26/72). However, almost all multiply recovered events (15/17) possess repeated sequences capable of accounting for the deletion endpoints. Similarily, over of all duplications recovered (5/7) display repeated endpoints. Single-base frameshifts are equally divided between A:T and G:C sites, in each case (−) 1 events occur 3-fold more frequently that (+)1 events. A comparative analysis of each mutational class recovered to lacI⁻ spectra available in a variety of DNA repair/metabolism-deficient strains is presented here in an attempt to assess possible contributions from chemical, physical and enzymic sources of damage.     

A new mutation rpoD800, affecting the sigma subunit of E. coli RNA polymerase is allelic to two other sigma mutants

Summary We have characterized a new mutation rpoD800 affecting the sigma gene of E. coli. Upon transfer to high temperature, a strain with the rpoD800 mutation ceases growth within 30 min. We find that this mutation renders sigma about 10-fold more thermolabile than the wild type sigma at 45°C in vitro. We have compared the temperature profile for inactivation of wild type and mutant sigma and find that the mutant inactivates at a temperature about 9° C lower than does the wild type.The chromosomal locus affected by rpoD800 is shown to be allelic to the locus affected by the spontaneous mutants ts285 and alt-1. All three mutations result in altered sigma and in altered growth at high temperature. We argue that the single locus affected is the structural gene for the sigma subunit of E. coli RNA polymerase.     

A novel plasmid-mediated DNA restriction-modification system in E. coli

R plasmids from 101 clinical isolates were transferred to E. coli J62 by conjugation and tested for the presence of R plasmid-mediated restriction-modification DNA systems. Thirty R plasmids were found to inhibit phage λ. vir development. Ten plasmids determined restriction modification system; nine of them proved identical with R.M. EcoRII. One transconjugant, E. coli J62 pLG74, was shown to have a restriction-modification system different from all the known R plasmid-mediated systems. Site-specific endonuclease has been isolated from E. coli J62 pLG74 which differed from all the known restriction endonucleases in the number of cleavage sites on phages λ, φX 174, virus SV40, plasmid pBR322 DNA molecules.     

The commonly-used DNA probe for diffusely-adherent Escherichia coli cross-reacts with a subset of enteroaggregative E. coli

Background  

The roles of diffusely-adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) in disease are not well understood, in part because of the limitations of diagnostic tests for each of these categories of diarrhoea-causing E. coli. A HEp-2 adherence assay is the Gold Standard for detecting both EAEC and DAEC but DNA probes with limited sensitivity are also employed.     

大肠杆菌严谨型RNA聚合酶的筛选及体内转录活性测定

利用选择性培养基筛选大肠杆菌自然突变菌株,经噬菌体P1转导和蛋白质互补试验,发现一株突变体(LCH001)的突变基因发生在编码RNA聚合酶β′亚基的rpoC基因上,经DNA序列分析,发现突变位点发生在第3406个碱基上,由G变成了T,导致编码的氨基酸由甘氨酸(GGT)变成半胱氨酸(TGT)。体内转录试验表明该突变RNA聚合酶转录严谨型启动子控制基因的活性显著降低,其β-半乳糖苷酶的活性是野生型菌株的18%,而转录非严谨型启动子控制基因的活性显著提高,其β-半乳糖苷酶的活性约是野生型菌株的5倍。研究结果对探讨RNA聚合酶结构与功能的关系以及RNA聚合酶在细菌严谨反应过程中的作用具有重要意义。     

Competition for the DNA template between RNA polymerase molecules from normal and phage-infected E. coli

Summary Preincubation of E. coli core RNA polymerase lacking sigma-factor with limiting amounts of T2-DNA markedly decreases subsequent synthesis of RNA by RNA polymerase holoenzyme. Hence, although the core binds to DNA more weakly than does the holoenzyme, it can actively compete with RNA polymerase for the DNA template.Both core RNA polymerase and holoenzyme from uninfected bacteria are effective in competition with RNA polymerase isolated from T2-infected cells. On the other hand the enzyme obtained from T2-infected cells compete weakly with RNA polymerase from E. coli. The incubation of bacterial core-enzyme with a supernatant protein fraction obtained from phage-infected bacteria lowers its ability to compete with normal RNA polymerase for DNA template.These results are discussed from the viewpoint that in certain cases the RNA polymerase itself can act as a kind of repressor, effecting negative regulation of RNA synthesis. The modification of core and formation of anti-sigma induced by bacteriophage could participate in such kind of regulation.     

Cloned truncated recA genes in E. coli

Summary Plasmids pMH1 and pDR1461, possessing the control region and 22% or 73% of the E. coli recA gene, conferred UV sensitivity to wild-type uvrA, and umuC bacteria. Sensitization was less in recA441 (tif-1) mutants and absent in lexA cells. Radiosensitization correlated with inhibition of recombinational repair, even through induced recA protein synthesis and recombination in Hfr matings were normal. Plasmids pMH1 and pDR1461 also prevented induction of some, but not all, SOS functions. Mutagenic reversion to tryptophan prototrophy and induced reactivation of UV-irradiated phage were eliminated, and the efficiency of lysogenic induction reduced. However, naladixic acid induced filamentous growth, mitomycin-C induced uvrA gene expression and post UV-irradiation DNA degradation control were little changed. Explanations of these effects are discussed which involve the presence of either truncated recA protein or multiple copies of the recA gene control sequence.A preliminary account of this work is presented in Chromosome Damage and Repair, edited by E. Seeberg and K. Klepper, to be published by Plenum Press     

Escherichia coli mutants with temperature-sensitive synthesis of DNA

Summary Seven mutants of E. coli with temperature-sensitive synthesis of DNA have been isolated. Synthesis of RNA, protein and DNA precursors does not appear to be directly affected. The mutants can be divided into at least two groups on the basis of their pattern of DNA synthesis, their ability to support phage growth at 41° and their genetic mapping.Mutants of the first group are heterogeneous in their pattern of DNA synthesis at 40°. Some mutants cease DNA synthesis abruptly upon transfer to 40° and any residual DNA synthesis is barely detectable. In others there is substantial residual synthesis at 40°. All these Group 1 mutants are alike, however, in that they support the growth of phage T4 but not Lambda at 41°. Two mutants with barely detectable residual DNA synthesis carry DNA mutations which have been mapped by P1 transduction and show about 72% linkage to the malB locus. It has not yet proved possible to map accurately the mutants showing substantial residual synthesis, and the possibility that these mutations are in a different gene(s) has not been excluded.A single mutant has been placed in a second group. Like some Group 1 mutants it synthesizes substantial amounts of DNA at 40° before synthesis stops. However, unlike them it supports the growth of T4 and Lambda at 41°. The DNA mutation maps near the leu locus. Certain properties of this mutant are consistent with the idea that initiation of DNA synthesis is temperature-sensitive in this strain.Adapted from a dissertation presented in partial fulfillment of the degree of Doctor of Philosophy. This investigation was supported in part by U.S. Public Health Services Grant 5-TO1-GM00829 from the National Institute of General Medical Sciences and in part by U.S.P.H.S. research grant GM12524.     

A mutation affecting L-serine and energy metabolism in E. coli K12

Summary The effects of a pleiotropic mutation ssd are described. This mutation results in decreased efficiency in the use of glucose and fructose as carbon source, inability to use succinate or to grow anaerobically, an alteration in the activity of enzymes responsible for the synthesis and degradation of L-serine, increased resistance to certain antibiotics, and a deficiency in proline transport. This mutation resembles various previously described mutations throught to affect energy coupling factor and is located in the same region of the chromosome. While the gene product affected by this mutation is still unidentified, it is clear that L-serine metabolism cannot be understood merely in terms of providing L-serine and its derivatives.     

大肠杆菌细胞DNA复制、修复和重组途径的衔接

以大肠杆菌为例围绕相关领域的研究动态进行分析和总结.DNA复制、损伤修复和重组过程的相互作用关系研究是当今生命科学研究的前沿和热点之一.越来越多的研究表明,在分子水平上,DNA复制、损伤修复和重组过程既彼此独立,又相互依存.上述途径可以通过许多关键蛋白质之间的相互作用加以协调和整合,并籍此使遗传物质DNA得到有效的维护和忠实的传递.需要指出的是,基于许多细胞内关键蛋白及其功能在生物界中普遍保守性的事实,相信来自大肠杆菌有关DNA复制、修复和重组之间的研究成果也会对相关真核生物的研究提供借鉴.