共查询到20条相似文献,搜索用时 31 毫秒
1.
T Yasuda K Morimatsu T Horii T Nagata H Ohmori 《Canadian Metallurgical Quarterly》1998,17(11):3207-3216
In Escherichia coli, the SOS response is induced upon DNA damage and results in the enhanced expression of a set of genes involved in DNA repair and other functions. The initial step, self-cleavage of the LexA repressor, is promoted by the RecA protein which is activated upon binding to single-stranded DNA. In this work, induction of the SOS response by the addition of mitomycin C was found to be prevented by overexpression of the dinI gene. dinI is an SOS gene which maps at 24.6 min of the E.coli chromosome and encodes a small protein of 81 amino acids. Immunoblotting analysis with anti-LexA antibodies revealed that LexA did not undergo cleavage in dinI-overexpressed cells after UV irradiation. In addition, the RecA-dependent conversion of UmuD to UmuD' (the active form for mutagenesis) was also inhibited in dinI-overexpressed cells. Conversely, a dinI-deficient mutant showed a slightly faster and more extensive processing of UmuD and hence higher mutability than the wild-type. Finally, we demonstrated, by using an in vitro reaction with purified proteins, that DinI directly inhibits the ability of RecA to mediate self-cleavage of UmuD. 相似文献
2.
3.
K Ishimori S Sommer A Bailone M Takahashi MM Cox R Devoret 《Canadian Metallurgical Quarterly》1996,264(4):696-712
A recA mutant (recA423; Arg169-->His), with properties that should help clarify the relationship between the biochemical properties of RecA protein and its two major functions, homologous genetic recombination and recombinational DNA repair, has been isolated. The mutant has been characterized in vivo and the purified RecA423 protein has been studied in vitro. The recA423 cells are nearly as proficient in conjugational recombination, transductional recombination, and recombination of lambda red- gam- phage as wild-type cells. At the same time, the mutant cells are deficient for intra-chromosomal recombination and nearly as sensitive to UV irradiation as a recA deletion strain. The cells are proficient in SOS induction, and results indicate the defect involves the capacity of RecA protein to participate directly in recombinational DNA repair. In vitro, the RecA423 protein binds to single-stranded DNA slowly, with an associated decline in the ATP hydrolytic activity. The RecA423 protein promoted a limited DNA strand exchange reaction when the DNA substrates were homologous, but no bypass of a short heterologous insert in the duplex DNA substrate was observed. These results indicate that poor binding to DNA and low ATP hydrolysis activity can selectively compromise certain functions of RecA protein. The RecA423 protein can promote recombination between homologous DNAs during Hfr crosses, indicating that the biochemical requirements for such genetic exchanges are minimal. However, the deficiencies in recombinational DNA repair suggest that the biochemical requirements for this function are more exacting. 相似文献
4.
DJ Farrell S Bilkhu LM Gibson L Cummings V Wadehra 《Canadian Metallurgical Quarterly》1997,41(2):251-260
A mathematical model for the development of the SOS signal in nucleotide-excision repair deficient Escherichia coli cells subjected to ultraviolet light irradiation is proposed, in which regions of single-stranded DNA (gaps) are created during replication of a damaged chromosome when the strand elongation stops at pyrimidine dimers. The concentration of single-stranded DNA of gaps as a function of time is obtained. The model for the interaction of the LexA and RecA proteins, a well-established key event in SOS regulation, is presented, resulting in a system of differential equations for the concentrations of LexA, RecA and activated RecA proteins. The simulated LexA protein kinetic curves agree with the experimental data for two excision repair deficient mutants: uvrA6 and dnaC28 uvrB(del), which is also a temperature-sensitive DNA replication initiation mutant. It is shown that the model can be used to quantitatively describe the kinetics of SOS response through the amount of the SOS signal (concentration of single-stranded DNA) in a cell as a function of time. 相似文献
5.
Two mutations in vaccinia virus topoisomerase I, K167D and G226N, have been characterized. SOS induction was observed in Escherichia coli expressing vaccinia topoisomerase I with either one of these mutations. The mutant enzymes were purified to homogeneity and compared with the wild type enzyme for relaxation activity and the partial activities of substrate binding, site-specific DNA cleavage and DNA religation to determine the mechanism of SOS induction. The K167D mutant enzyme had reduced binding affinity for the DNA substrate with a Kapp that was 10-fold higher than wild type. Nevertheless, in reactions with high enzyme concentration, its substrate cleavage activity was 90% that of wild type. The G226N mutant enzyme had virtually wild type binding and cleavage activities. However, intermolecular religation by these two mutants were observed to be significantly reduced. The cleavage complexes formed with the K167D and G226N mutants were more stable to high salt than the wild type cleavable complex. We propose that these mutants in vivo induce the SOS response in E. coli due to the shift of topoisomerase cleavage-religation equilibrium towards cleavage and increased stability of the cleavage complex. The mutation thus has a similar effect as the topoisomerase-targeting inhibitors that turn topoisomerases into DNA damaging agents. 相似文献
6.
In addition to its critical role in genetic recombination, the Escherichia coli RecA protein plays a pivotal role in SOS-induced mutagenesis. This role can be separated genetically into three steps: (i) depression of the SOS regulon by mediating the posttranslational cleavage of the LexA repressor, (ii) activation of UmuD'-like proteins by mediating cleavage of the UmuD-like proteins, and (iii) a direct step, possibly to interact with and to target the Umu-like mutagenesis proteins to lesions in DNA. We have analyzed RecA's third role biochemically using protein affinity chromatography and an agarose-based DNA mobility-shift assay. RecA730 protein from a crude cell extract was specifically retained on UmuD and UmuD' protein affinity columns, suggesting that these proteins physically interact. Normally, neither UmuD nor UmuD' shows any affinity for DNA. In the presence of RecA protein, however, UmuD and UmuD' were targeted to DNA. RecA1730 protein, which is defective for UmuD' but proficient for MucA'-promoted mutagenesis, showed a dramatically reduced capacity to target UmuD' to DNA but was able to target a significant portion of MucA' to DNA. These data support the suggestion that the direct role of RecA protein in SOS-induced mutagenesis is to interact with and target the Umu-like mutagenesis proteins to DNA. 相似文献
7.
BACKGROUND: The RAD51 gene of Saccharomyces cerevisiae is homologous to the Escherichia coli recA gene and plays a key role in genetic recombination and DNA double-strand break repair. To construct an improved experimental system of homologous recombination in higher eukaryotes, we have chosen the South African clawed frog, Xenopus laevis, whose egg extracts might be useful for the in vitro studies. We identified and characterized a Xenopus homologue of RAD51 gene, the XRAD51.1. RESULTS: Recombinant XRad51.1 was expressed in E. coli. The purified XRad51.1 protein showed ssDNA-dependent ATPase activity and promoted the DNA strand exchange reaction between two 55-mer oligonucleotides. The binding stoichiometry of XRad51.1 to ssDNA was determined by fluorescence of poly(d epsilonA), a chemically modified poly(dA), and was found to be about six bases/XRad51.1 monomer in a nucleoprotein filament, a similar value to E. coli RecA protein. The kinetics of the fluorescence change of poly(d epsilonA) after XRad51.1 binding in the presence of ATP was significantly different from that observed with RecA protein. The affinity of XRad51.1 to ssDNA in the presence of ATP was higher than that of RecA protein, and the dissociation of the XRad51.1-ssDNA complex was slower than the RecA-ssDNA complex. CONCLUSIONS: Purified recombinant XRad51.1 protein promoted the strand exchange between short DNA molecules. While the binding stoichiometry of XRad51.1 protein to ssDNA was identical to that of the RecA protein, XRad51.1 has a significantly higher affinity and binding stability to ssDNA than did the RecA protein in the presence of ATP. 相似文献
8.
WM Rehrauer I Bruck R Woodgate MF Goodman SC Kowalczykowski 《Canadian Metallurgical Quarterly》1998,273(49):32384-32387
The RecA, UmuC, and UmuD' proteins are essential for error-prone, replicative bypass of DNA lesions. Normally, RecA protein mediates homologous pairing of DNA. We show that purified Umu(D')2C blocks this recombination function. Biosensor measurements establish that the mutagenic complex binds to the RecA nucleoprotein filament with a stoichiometry of one Umu(D')2C complex for every two RecA monomers. Furthermore, Umu(D')2C competitively inhibits LexA repressor cleavage but not ATPase activity, implying that Umu(D')2C binds in or proximal to the helical groove of the RecA nucleoprotein filament. This binding reduces joint molecule formation and even more severely impedes DNA heteroduplex formation by RecA protein, ultimately blocking all DNA pairing activity and thereby abridging participation in recombination function. Thus, Umu(D')2C restricts the activities of the RecA nucleoprotein filament and presumably, in this manner, recruits it for mutagenic repair function. This modulation by Umu(D')2C is envisioned as a key event in the transition from a normal mode of genomic maintenance by "error-free" recombinational repair, to one of "error-prone" DNA replication. 相似文献
9.
D Morgan L Turnpenny J Goodship W Dai K Majumder L Matthews A Gardner G Schuster L Vien W Harrison FF Elder M Penman-Splitt P Overbeek T Strachan 《Canadian Metallurgical Quarterly》1998,20(2):149-156
A DNA fragment containing the recA gene of Gluconobacter oxydans was isolated and further characterized for its nucleotide sequence and ability to functionally complement various recA mutations. When expressed in an Escherichia coli recA host, the G. oxydans recA protein could efficiently function in homologous recombination and DNA damage repair. The recA gene's nucleotide sequence analysis revealed a protein of 344 amino acids with a molecular mass of 38 kDa. We observed an E. coli-like LexA repressor-binding site in the G. oxydans recA gene promoter region, suggesting that a LexA-like mediated response system may exist in G. oxydans. The expression of G. oxydans recA in E. coli RR1, a recA+ strain, surprisingly caused a remarkable reduction of the host wild-type recA gene function, whereas the expression of both Serratia marcescens recA and Pseudomonas aeruginosa recA gene caused only a slight inhibitory effect on function of the host wild-type recA gene product. Compared with the E. coli RecA protein, the identity of the amino acid sequence of G. oxydans RecA protein is much lower than those RecA proteins of both S. marcescens and Pseudomonas aeruginosa. This result suggests that the expression of another wild-type RecA could interfere with host wild-type recA gene's function, and the extent of such an interference is possibly correlated to the identity of the amino acid sequence between the two classes of RecA protein. 相似文献
10.
The Res subunits of the type III restriction-modification enzymes share a statistically significant amino acid sequence similarity with several RNA and DNA helicases of the so-called DEAD family. It was postulated that in type III restriction enzymes a DNA helicase activity may be required for local unwinding at the cleavage site. The members of this family share seven conserved motifs, all of which are found in the Res subunit of the type III restriction enzymes. To determine the contribution, if any, of these motifs in DNA cleavage by EcoPI, a type III restriction enzyme, we have made changes in motifs I and II. While mutations in motif I (GTGKT) clearly affected ATP hydrolysis and resulted in loss of DNA cleavage activity, mutation in motif II (DEPH) significantly decreased ATP hydrolysis but had no effect on DNA cleavage. The double mutant R.EcoPIK90R-H229K showed no significant ATPase or DNA restriction activity though ATP binding was not affected. These results imply that there are at least two ATPase reaction centres in EcoPI restriction enzyme. Motif I appears to be involved in coupling DNA restriction to ATP hydrolysis. Our results indicate that EcoPI restriction enzyme does not have a strand separation activity. We suggest that these motifs play a role in the ATP-dependent translocation that has been proposed to occur in the type III restriction enzymes. 相似文献
11.
MA Rab MK Bile MM Mubarik H Asghar Z Sami S Siddiqi AS Dil MA Barzgar MA Chaudhry MI Burney 《Canadian Metallurgical Quarterly》1997,57(2):151-157
Following the digestion of chromosomal DNA of Deinococcus radiodurans with a restriction enzyme a partial genomic library was constructed using lambda phage as a vector. A phage clone whose DNA can complement the deficiency in a radiation-sensitive mutant of D. radiodurans was isolated. Following the subcloning using phasmid vector, a hybrid plasmid containing 1.2 kb inserted DNA was obtained. After the determination of nucleotide sequence, the deduced amino acid sequence showed close homology to RuvB protein of Escherichia coli; approximately 81% of the amino acids (310 residues in total) was homologous (152 were identical and 100 amino acids were similar). The putative protein has a conserved ATP binding domain characteristic of DNA helicases. However, we could not find an SOS promoter and ORF for RuvA protein in the sequence upstream of ruvB in contrast to the E. coli homologue. The mutant was transformed with exogenous DNA at the same rate as the wild-type cells, but it was moderately sensitive to UV, gamma-rays and to interstrand cross-linking reagents. 相似文献
12.
Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription 总被引:1,自引:0,他引:1
T Kogoma 《Canadian Metallurgical Quarterly》1997,61(2):212-238
13.
Crystal structure of a DExx box DNA helicase 总被引:1,自引:0,他引:1
HS Subramanya LE Bird JA Brannigan DB Wigley 《Canadian Metallurgical Quarterly》1996,384(6607):379-383
There are a wide variety of helicases that unwind helical DNA and RNA substrates. The twelve helicases that have been identified in Escherichia coli play a role in almost all cellular processes involving nucleic acids. We have solved the crystal structure of a monomeric form of a DNA helicase from Bacillus stearothermophilus, alone and in a complex with ADP, at 2.5 and 2.9 A resolution, respectively. The enzyme comprises two domains with a deep cleft running between them. The ATP-binding site, which is situated at the bottom of this cleft, is formed by motifs that are conserved across the superfamily of related helicases. Unexpected structural homology with the DNA recombination protein, RecA, suggests how ATP binding and hydrolysis may drive conformational changes of the enzyme during catalysis, and implies that there is a common mechanism for all helicases. 相似文献
14.
E Dahan-Grobgeld Z Livneh AF Maretzek S Polak-Charcon Z Eichenbaum H Degani 《Canadian Metallurgical Quarterly》1998,273(46):30232-30238
Early metabolic events in Escherichia coli exposed to nalidixic acid, a topoisomerase II inhibitor and an inducer of the SOS system, were investigated by in vivo NMR spectroscopy, a technique that permits monitoring of bacteria under controlled physiological conditions. The energetics of AB1157 (wild type) and of its isogenic, SOS-defective mutants, recBC, lexA, and DeltarecA, were studied by 31P and 19F NMR before, during, and after exposure to nalidixic acid. The content of the NTP in E. coli embedded in agarose beads and perfused at 36 degreesC was found to be 4.3 +/- 1.1 x 10(-18) mol/cell, yielding a concentration of approximately 2.7 +/- 0.7 mM. Nalidixic acid induced in the wild type and mutants a rapid 2-fold increase in the content of the NTP, predominantly ATP. This induction did not involve synthesis of uracil derivatives or breakdown of RNA and caused cell proliferation to stop. Removal of nalidixic acid after 40 min of treatment rescued the cells and resulted in a decrease of ATP to control levels and resumption of proliferation. However, in DeltarecA cells, which were more sensitive to the activity of the drug, ATP elevation could not be reversed, and ATP content continued to increase faster than in control cells. The results ruled out association between the elevation of ATP and the induction of the SOS system and suggested involvement of a process reminiscent of apoptosis in the stimulation of ATP synthesis. Thus, the presence of the RecA protein was found to be essential for reversing the ATP increase and cell rescue, possibly by its function in repair of DNA damage. 相似文献
15.
The recA gene of a thermophilic eubacterial strain, Thermus thermophilus (T.th.) HB8, was cloned from a genomic DNA library by Southern hybridization using a gene-internal fragment amplified by the polymerase chain reaction (PCR) method as the probe. The gene encoded a 36 kDa polypeptide whose amino acid sequence showed 61% identity with that of the Escherichia coli RecA protein. Characteristic amino acid changes between the two RecA proteins were found. In the amino acid composition of the T.th. RecA protein, the number of Pro residues was increased, the number of Cys residues was decreased, and Lys residues were replaced by Arg, Asp by Glu, Thr by Val, and Ile by Val or Leu. These changes are supposed to stabilize the native protein conformation against heat denaturation. The amino acid residues in the nucleotide binding site of the protein and in the protein-protein interaction site responsible for the oligomer formation were well conserved. The T.th. recA gene has the ability to complement the ultraviolet light (UV) sensitivity of a E. coli recA deletion mutant. Thus, the thermophilic bacterium has a RecA protein whose function will be common to the E. coli RecA protein. 相似文献
16.
17.
We have developed a new assay to characterize the double-stranded DNA (dsDNA) binding properties of RecA protein. This assay is based on measurement of changes in the fluorescence of a 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complex upon RecA protein binding. The binding of RecA protein to a complex of DAPI and dsDNA results in displacement of the bound DAPI, producing a decrease in the observed fluorescence. DAPI displacement is dependent on both RecA protein and ATP; dATP and, to a lesser extent, UTP and dCTP also support the DAPI displacement reaction, but dGTP, GTP, dITP and TTP do not. Binding stoichiometry for the RecA protein-dsDNA complex measured by DAPI displacement is 3 bp per RecA protein monomer in the presence of ATP. These results, taken together with data for mutant RecA proteins, suggest that this DAPI displacement assay monitors formation of the high affinity DNA binding state of RecA protein. Since this state of RecA protein defines the form of the nucleoprotein filament that is active in DNA strand exchange, these findings raise the possibility that the RecA protein-dsDNA filament may possess a homologous pairing capacity. 相似文献
18.
19.
The Escherichia coli RecA protein has served as a model for understanding protein-catalyzed homologous recombination, both in vitro and in vivo. Although RecA proteins have now been sequenced from over 60 different bacteria, almost all of our structural knowledge about RecA has come from studies of the E. coli protein. We have used electron microscopy and image analysis to examine three different structures formed by the RecA protein from the thermophilic bacterium Thermus aquaticus. This protein has previously been shown to catalyze an in vitro strand exchange reaction at an optimal temperature of about 60 degrees C. We show that the active filament formed by the T. aquaticus RecA on DNA in the presence of a nucleotide cofactor is extremely similar to the filament formed by the E. coli protein, including the extension of DNA to a 5.1-A rise per base pair within this filament. This parameter appears highly conserved through evolution, as it has been observed for the eukaryotic RecA analogs as well. We have also characterized bundles of filaments formed by the T. aquaticus RecA in the absence of both DNA and nucleotide cofactor, as well as hexameric rings of the protein formed under all conditions examined. The bundles display a very large plasticity of mass within the RecA filament, as well as showing a polymorphism in filament-filament contacts that may be important to understanding mutations that affect surface residues on the RecA filament. 相似文献
20.
Two site-directed mutants of Escherichia coli DNA helicase II (UvrD) were constructed to examine the functional significance of motif VI in a superfamily I helicase. Threonine 604 and arginine 605, representing two of the most highly conserved residues in motif VI, were replaced with alanine, generating the mutant alleles uvrD-T604A and uvrD-R605A. Genetic complementation studies indicated that UvrD-T604A, but not UvrD-R605A, functioned in methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair. Both mutant enzymes were purified and single-stranded DNA (ssDNA)-stimulated ATP hydrolysis, duplex DNA unwinding, and ssDNA binding were studied in the steady-state and compared to wild-type UvrD. UvrD-T604A exhibited a serious defect in ssDNA binding in the absence of nucleotide. However, in the presence of a non-hydrolyzable ATP analog, DNA binding was only slightly compromised. Limited proteolysis experiments suggested that UvrD-T604A had a "looser" conformation and could not undergo conformational changes normally associated with ATP binding/hydrolysis and DNA binding. UvrD-R605A, on the other hand, exhibited nearly normal DNA binding but had a severe defect in ATP hydrolysis (kcat=0.063 s-1 compared to 162 s-1 for UvrD). UvrD-T604A exhibited a much less severe decrease in ATPase activity (kcat=8.8 s-1). The Km for ATP for both mutants was not significantly changed. The results suggest that residues within motif VI of helicase II are essential for multiple biochemical properties associated with the enzyme and that motif VI is potentially involved in conformational changes related to the coupling of ATPase and DNA binding activities. 相似文献