质粒的复制特性和整合后在发动大肠杆菌染色体复制中对recA基因的依赖性的研究

大肠杆菌的温度敏感的DNA复制发动突变型dnaA46在42℃中不能生长而形成菌落,质粒或噬菌体的整合使它能生长。前文[2]报道了F质粒整合后所发动的染色体复制依赖于recA基因。本文报道5种质粒和2种噬菌体整合后所发动的染色体复制对recA基因的依赖性的研究结果。实验结果说明,依赖与否和它们在游离状态中复制方向无关,从而否定了recA基因的作用是把质粒或噬菌体的游离状态的单向复制发动机构改变为整合状态的双向复制发动饥构这一假设。     

带有特定染色体片段的mini-F质粒所发动的染色体复制对于recA基因的依赖性

构建了一些带有大肠杆菌的特定染色体片段的mini-F质粒,使它们通过同源重组整合在dnaA46细菌的染色体的预定位置上,然后测定整合抑制菌株(sin)在40℃中染色体复制对recA基因的依赖性。实验结果说明,Sin菌株对recA基因的依赖性决定在质粒的整合位置。整合在oriC近旁的Sin菌株不依赖于recA基因;整合在oriC和terC中间的只在丰富培养基上是依赖的;整合在rerC附近的在不丰富的培养基上也依赖于recA基因。     

大肠杆菌K-12dnaA突变型的R6K整合抑制菌株的多起点染色体复制

大肠杆菌K-12的温度敏感复制发动缺陷突变(dnaA46)菌株LC381不能在42℃中进行染色体复制。在42℃中选取R6K质粒整合抑制菌株,用标记频率测定法测得这一菌株在30℃中染色体复制从正常的复制起点起始,在42℃中则从另外三个起点起始,其中两个曾见报道,把重组突变recA56引入这一菌株,发现由接近正常复制起点起始的染色体复制不受recA突变的影响,由接近正常复制终点起始的染色体复制则受到阻碍,说明由这一位置起始的染色体复制依赖于recA基因。这一实验结果和我们的其他报道相符。     

带有IS1的mini—F质粒所发动的染色体复制对于recA基因的依赖性

我们曾报道整合的F′质粒所发动的大肠杆菌染色体复制依赖于recA基因,而整合的F质粒则不。构建带有IS1的mini-F质粒,它们的复制起点分别来自F或F′质粒。这些质粒的整合抑制菌株中都有约20％是recA依赖的,不管这一mini-F质粒的复制起点来自F或F′质粒,也不管这一质粒在游离状态中的复制方向是单向或双向。实验结果说明,质粒的整合位置是决定由整合质粒所发动的染色体复制对recA基因的依赖性的主要因素。     

大肠杆菌中整合的F′质粒带动细菌染色体复制需要recA基因

大肠杆菌的dnaA46突变能被F′质粒整合抑制。整合抑制的菌林(Sin菌株)在通过转导引入了recA56突变后又变得不能在40℃中生长。标记转移、吖啶橙敏感性,F′质粒消除和mini-染色体质粒转化等实验说明,Sin菌株中F′质粒始终处于整合状态,并且在40℃中细菌染色体的复制由整合状态的F′质粒所带动。比较了Sin recA~ 和Sin recA~-菌株在不同温度中的DNA、蛋白质的生物合成情况。实验结果说明recA基因在DNA复制过程中起作用。前人的工作证明了recA基因在DNA重组和DHA损伤应急修复(SOS)过程中是一个关键的基因。本文的工作为recA基因的功能提供了新的认识。     

大肠杆菌中整合状态的F′质粒复制起点的克隆和分析

用标记获救法克隆了整合状态的F′质粒的复制起点,证明了由这一复制起点构成的mini-F质粒在不亲和性和对吖啶橙的敏感性方面和自主状态的F′质粒都没有不同。对这一复制起点和来自自主状态的F质粒的复制起点进行了亚克隆,并作限制性内切酶酶切分析比较,没有发现两者在结构上有差异。本文的结果提示,F质粒和F′质粒在发动染色体复制中对recA基因的依赖性的不同,可能与质粒整合在染色体上的位置不同有关。     

拟南芥SUA41基因的表达和功能分析

植物的开花受多条途径的控制, 其中包括光周期途径、春化途径、赤霉素途径、自主途径和温敏途径。SUA41(SUMO substrate in Arabidopsis 41)是本实验室筛选到的、SUMO(Small ubiquitin modifier)的潜在底物, 并且前人的研究发现它参与自主途径的开花调节, 但其对开花时间的调节机制没有详细报道。文章对SUA41基因的表达、sua41突变体对不同环境条件的反应以及SUA41对开花时间调节的可能机制进行初步分析。结果显示, 与野生型相比, sua41突变体在常温或低温、长日或者短日条件下均为早花, 并且在低温和常温下的开花时间没有太大差别。过表达SUA41能够恢复sua41突变体的早花表型。SUA41基因在拟南芥的幼苗、根、茎、叶和花以及各个植物发育阶段都有表达, 说明SUA41基因是一个组成型表达基因。SUA41基因的表达与GA处理无关, 长日低温条件能够诱导SUA41基因的表达, 且在温敏途径突变体fve和fca中SUA41基因的表达量减少。与野生型比较, sua41突变体中CO基因的mRNA表达量没有明显变化, FT和SOC1基因表达量增加且FT增加幅度更大, FLC的mRNA表达量减少。结果表明SUA41基因虽然在自主途径中起作用, 但主要在温敏途径中参与拟南芥开花时间调节。     

recA基因的克隆及其在λ溶原菌中的生物学功能

通过一对自行设计的引物,以E. coli染色体DNA为模板进行PCR扩增,获得完整的recA基因.将PCR产物和pUC18在体外进行连接后,并分别转入E. coli DH5α和E.coli k12(λ+),筛选出含重组质粒(pUR4)的转化子.在诱导条件和非诱导条件下,分别测定recA基因在不同转化子中的生物学功能.结果表明recA基因在溶原菌中表现出明显的生理功能,能使λ原噬菌体从溶原状态进入裂解循环.这种重组质粒将在进一步研究λ原噬菌体的诱导机理以及受紫外辐射损伤细胞的修复作用等方面成为有效的工具.     

recA基因的克隆及其在λ溶原菌中的生物学功能

通过一对自行设计的引物,以E．coli染色体DNA为模板进行PCR扩增,获得完整的recA基因。将PCR产物和pUCl8在体外进行连接后,并分别转入E.coli DH5α和E.coli k12(λ^ ),筛选出含重组质粒(pUR4)的转化子。在诱导条件和非诱导条件下,分别测定recA基因在不同转化子中的生物学功能。结果表明recA基因在溶原菌中表现出明显的生理功能,能使九原噬菌体从溶原状态进入裂解循环。这种重组质粒将在进一步研究九原噬菌体的诱导机理以及受紫外辐射损伤细胞的修复作用等方面成为有效的工具。     

耐辐射奇球菌recA基因的克隆、表达及其对recA缺损大肠杆菌辐射抗性的影响

将耐辐射奇球菌(Deinococcus radiodurans)recA基因克隆到表达质粒pET15b中,并在Escherichia coli HMS中高效表达了可溶性的RecA重组蛋白。同时将recA基因通过穿梭质粒pRADZ3导入recA缺损E.coli TG2细胞中,Western印迹实验显示RecA蛋白能够在不需要诱导剂IPTG的条件下稳定表达。辐射抗性实验表明,D.radiodurans的recA基因在E.coli细胞中的表达能够完全补偿recA缺损E.coli辐射抗性能力。     

Insertion of inverted Ter sites into the terminus region of the Escherichia coli chromosome delays completion of DNA replication and disrupts the cell cycle

To investigate the co-ordination between DNA replication and cell division, we have disrupted the DNA replication cycle of Escherichia coli by inserting inverted Ter sites into the terminus region to delay completion of the chromosome. The inverted Ter sites (designated Inv Ter :: spc ^r) were initially inserted into the chromosome of a Δ tus strain to allow unrestrained chromosomal replication. We then introduced a functional tus gene by transforming the Inv Ter :: spc ^r strain with a plasmid carrying the tus gene under control of an arabinose-inducible promoter. In the presence of 0.2% arabinose, the cells formed long filaments, suggesting that activation of the inverted Ter sites by Tus arrested DNA replication and delayed the onset of cell division. Induction of sfiA , a gene in the SOS regulon, was observed following arrest of DNA replication; however, when a sfiB114 allele was introduced into Inv Ter :: spc ^r strain, long filaments were still formed, suggesting that the sfi -independent pathway also caused filamentation. Either recA :: cam ^r or lexA3 alleles suppressed filamentation when introduced in the Inv Ter strain. Interestingly, in both the recA :: cam ^r and lexA3 mutants, virtually all cells had a nucleoid, suggesting that cell division was proceeding even though DNA replication was not complete. These results suggest that DNA replication and cell division are uncoupled when recA is inactivated or when genes repressed by LexA cannot be induced.     

Bacteriophage P2: recombination in the superinfection preprophage state and under replication control by phage P4

Genetic crosses (mixed infection, lytic cycle) with bacteriophage P2 are known to give extremely low recombination frequencies, and these are unaffected by the recA status of the host bacterium. We now show the following: (1) the satellite bacteriophage P4, which interacts with P2 in a number of ways, but is quite different from it in terms of DNA replication and its control, is clearly dependent on the host recA+ function for recombination; (2) a chimeric phage (Lindqvist's P2/P4 Hy19), in which P2 replication early genes have been replaced by those of P4, recombines in a recA+-dependent manner; (3) immunity-sensitive P2 phages, in mixed infections of P2-immune bacteria, and hence blocked in their replication, recombine in a recA+-dependent manner; (4) an analysis of the distribution of exchanges based on a simple model confirms that in mixed infections of sensitive cells (where P2 is actively multiplying) recombinational exchanges tend to be statistically clustered in a segment of the chromosome containing the origin of replication, and also shows that, under conditions in which P2 DNA replication is blocked, the distribution of exchanges correlates well with the physical distances between markers on the P2 DNA.     

Roles of Ruva, Ruvc and Recg Gene Functions in Normal and DNA Damage-Inducible Replication of the Escherichia Coli Chromosome

Induction of the SOS response in Escherichia coli activates normally repressed DNA replication which is termed inducible stable DNA replication (iSDR). We previously demonstrated that initiation of iSDR requires the products of genes, such as recA, recB and recC, that are involved in the early stages of homologous recombination. By measuring the copy number increase of the origin (oriM1) region on the chromosome, we show, in this study, that initiation of iSDR is stimulated by mutations in the ruvA, ruvC and recG genes which are involved in the late stages of homologous recombination. Continuation of iSDR, on the other hand, is inhibited by these mutations. The results suggest that Holliday recombination intermediates, left on the chromosome due to abortive recombination, arrest replication fork movement. Low levels of iSDR and sfiA (sulA) gene expression were also observed in exponentially growing ruvA, ruvC and recG mutants, suggesting that the SOS response is chronically induced in these mutants. We propose that replication forks are arrested in these mutants, albeit at a low frequency, even under the normal (uninduced) conditions.     

Loss of RecA function affects the ability of Escherichia coli to maintain recombinant plasmids containing a Ter site.

In the Escherichia coli chromosome, DNA replication forks arrested by a Tus-Ter complex or by DNA damage are reinitiated through pathways that involve RecA and numerous other recombination functions. To examine the role of recombination in the processing of replication forks arrested by a Tus-Ter complex, the requirements for recombination-associated gene products were assessed in cells carrying Ter plasmids, i.e., plasmids that contain a Ter site oriented to block DNA replication. Of the E. coli recombination functions tested, only loss of recA conferred an observable phenotype on cells containing a Ter plasmid, which was inefficient transformation and reduced ability to maintain a Ter plasmid when Tus was expressed. Given the current understanding of replication reinitiation, the simplest explanation for the restriction of Ter plasmid maintenance was a reduced ability to restart plasmid replication in a recA tus(+) background. However, we were unable to detect a difference in the efficiency of replication arrest by Tus in recA-proficient and recA-deficient cells, which suggests that the inability to restart arrested replication forks is not the cause of the restriction on growth, but is due to an additional function provided by RecA. Other explanations for restriction of Ter plasmid maintenance were examined, including plasmid multimerization, plasmid rearrangements, and copy number differences. The most likely cause of the restriction on Ter plasmid maintenance was a reduced copy number in recA cells that was detected when the copy number was measured in relation to an external control. Possibly, loss of RecA function leads to improper processing of replication forks arrested at a Ter site, leading to the generation of degradation-prone substrates.     

Replication mutations differentially enhance RecA-dependent and RecA-independent recombination between tandem repeats in Bacillus subtilis

We have studied DNA recombination between 513 bp tandem direct repeats present in a kanamycin resistance gene inserted in the Bacillus subtilis chromosome. Tandem repeat deletion was not significantly affected by a recA mutation. However, recombination was stimulated by mutations in genes encoding replication proteins, including the primosomal proteins DnaB, DnaD and the DnaG primase, the putative DNA polymerase III subunits PolC, DnaN and DnaX, as well as the DNA polymerase DnaE. Hyper-recombination was found to be dependent on RecA in the dnaE, dnaN and dnaX mutants, whereas the dnaG and dnaD mutants stimulated recombination independently of RecA. Altogether, these data show that both RecA-dependent and RecA-independent mechanisms contribute to recombination between tandem repeats in B. subtilis and that both types of recombination are stimulated by replication mutations.     

SOS induction of the gene sulA is partly inhibited in Escherichia coli K12 cells overproducing the RecA protein

A study was made of the SOS induction of the gene sulA of Escherichia coli K12 in relation to the gene dosage of the gene recA. In experiments the sulA::lacZ fusion strain PQ37 and derivatives of PQ37 with the multi-copy plasmids pDR1453 or pBR322 were used. The SOS response was induced with nitrofurantoin, SOS induction of the gene sulA was determined on the basis of the amount of beta-galactosidase synthesized, i.e. by the SOS chromotest (Quillardet et al., 1982a). It was found in this work that cells with the plasmid pDR1453, which contain the gene recA of E. coli K12 (Sancar and Rupp, 1979), have a decreased SOS induction of the gene sulA. Cells with the plasmid pBR322 do not exhibit this decrease. Inactivation of the gene recA in the plasmid pDR1453 with preservation of the functional gene recA in the chromosome leads to a restoration of 'standard' SOS induction of the gene sulA. The results show that the amount of the gene product of the gene recA affects the SOS induction of the gene sulA.     

Tus-mediated arrest of DNA replication in Escherichia coli is modulated by DNA supercoiling

In the absence of RecA, expression of the Tus protein of Escherichia coli is lethal when ectopic Ter sites are inserted into the chromosome in an orientation that blocks completion of chromosome replication. Using this observation as a basis for genetic selection, an extragenic suppressor of Tus-mediated arrest of DNA replication was isolated with diminished ability of Tus to halt DNA replication. Resistance to tus expression mapped to a mutation in the stop codon of the topA gene (topA869), generating an elongated topoisomerase I protein with a marked reduction in activity. Other alleles of topA with mutations in the carboxyl-terminal domain of topoisomerase I, topA10 and topA66, also rendered recA strains with blocking Ter sites insensitive to tus expression. Thus, increased negative supercoiling in the DNA of these mutants reduced the ability of Tus-Ter complexes to arrest DNA replication. The increase in superhelical density did not diminish replication arrest by disrupting Tus-Ter interactions, as Tus binding to Ter sites was essentially unaffected by the topA mutations. The topA869 mutation also relieved the requirement for recombination functions other than recA to restart replication, such as recC, ruvA and ruvC, indicating that the primary effect of the increased negative supercoiling was to interfere with Tus blockage of DNA replication. Introduction of gyrB mutations in combination with the topA869 mutation restored supercoiling density to normal values and also restored replication arrest at Ter sites, suggesting that supercoiling alone modulated Tus activity. We propose that increased negative supercoiling enhances DnaB unwinding activity, thereby reducing the duration of the Tus-DnaB interaction and leading to decreased Tus activity.     

recA Gene dependence of replication of the Escherichia coli chromosome initiated by plasmid pUC13 integrated at predetermined sites

Summary Plasmid pUC13 was used to clone DNA fragments of known sites from the chromosome of Escherichia coli. Each chimeric plasmid was introduced individually into the same dnaA46 mutant strain LC381 and suppressive integration (Sin) strains were selected. By means of cotransduction the null mutation recA56 was then introduced into each Sin strain and growth of each recA56 derivative at 42° C was scored. Strains that failed to grow at 42° C depended upon the recA gene for replication. Three factors were shown to limit the viability of LC381 harboring different chimeric plasmids and affect the degree of recA gene dependence of chromosome replication in the Sin strains at 42° C. It is suggested that these three constraints are the consequence of the organization of the E. coli chromosome, particularly the unique ability of terC to retard the progression of replication forks. Two classes of hypotheses concerning the function of the recA gene are considered.