共查询到20条相似文献,搜索用时 15 毫秒
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Kow YW Imhoff B Weiss B Hung DC Hindoyan AA Story RM Goodman SD 《Nucleic acids research》2007,35(19):6672-6680
HU is one of the most abundant DNA binding proteins in Escherichia coli. We find that it binds strongly to DNA containing an abasic (AP) site or tetrahydrofuran (THF) (apparent Kd ≈50 nM). It also possesses an AP lyase activity that cleaves at a deoxyribose but not at a THF residue. The binding and cleavage of an AP site was observed only with the HUαβ heterodimer. Site-specific mutations at K3 and R61 residues led to a change in substrate binding and cleavage. Both K3A(α)K3A(β) and R61A(α)R61A(β) mutant HU showed significant reduction in binding to DNA containing AP site; however, only R61A(α)R61A(β) mutant protein exhibited significant loss in AP lyase activity. Both K3A(α)K3A(β) and R61K(α)R61K(β) showed slight reduction in AP lyase activities. The function of HU protein as an AP lyase was confirmed by the ability of hupA or hupB mutations to further reduce the viability of an E. coli dut(Ts) xth mutant, which generates lethal AP sites at 37°C; the hupA and hupB derivatives, respectively, had a 6-fold and a 150-fold lower survival at 37°C than did the parental strain. These data suggest, therefore, that HU protein plays a significant role in the repair of AP sites in E. coli. 相似文献
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Immotile phenotype of an Escherichia coli mutant lacking the histone-like protein HU 总被引:1,自引:0,他引:1
Satoshi Nishida Tohru Mizushima Takeyoshi Miki Kazuhisa Sekimizu 《FEMS microbiology letters》1997,150(2):297-301
The histone-like protein HU in Escherichia coli are encoded by the hupA and hupB genes. A hupA-hupB double deletion mutant has now been shown to express an immotile phenotype. The motility of hupA or hupB single mutants was similar to that of wild-type cells. SDS-polyacrylamide gel electrophoresis revealed that the amount of flagellin in the hupA-hupB double deletion mutant was markedly reduced compared with the wild-type strain, suggesting that the immotile phenotype of the double deletion mutant is caused by a loss of flagella. 相似文献
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Participation of the histone-like protein HU and of IHF in minichromosomal maintenance in Escherichia coli 总被引:8,自引:0,他引:8
The closely related Escherichia coli genes, hupA, hupB, himA and himD (hip), encode the bacterial histone-like protein subunits, HU-2, HU-1, IHF chi and IHF beta, respectively. We report here that E. coli minichromosomes [plasmids (2.7-12.2 kb) with oriC] carrying the intact mioC region were unable to transform mutants deficient in both HU and integration host factor (IHF), whereas they could transform mutants deficient in either HU or IHF as efficiently as the wild-type strain. Minichromosomes carrying a deletion of the proximal part of mioC or a DnaA box just upstream from mioC could not transform cells deficient in IHF, but could transform cells deficient in HU. These results suggested that HU and IHF participate in minichromosomal replication from oriC in E. coli. 相似文献
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Closely opposed lesions form a unique class of DNA damage that is generated by ionizing radiation. Improper repair of closely opposed lesions could lead to the formation of double strand breaks that can result in increased lethality and mutagenesis. In vitro processing of closely opposed lesions was studied using double-stranded DNA containing a nick in close proximity opposite to a dihydrouracil. In this study we showed that HU protein, an Escherichia coli DNA-binding protein, has a role in the repair of closely opposed lesions. The repair of dihydrouracil is initiated by E. coli endonuclease III and processed via the base excision repair pathway. HU protein was shown to inhibit the rate of removal of dihydrouracil by endonuclease III only when the DNA substrate contained a nick in close proximity opposite to the dihydrouracil. In contrast, HU protein did not inhibit the subsequent steps of the base excision repair pathway, namely the DNA synthesis and ligation reactions catalyzed by E. coli DNA polymerase and E. coli DNA ligase, respectively. The nick-dependent selective inhibition of endonuclease III activity by HU protein suggests that HU could play a role in reducing the formation of double strand breaks in E. coli. 相似文献
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Hong Ha Nguyen Claire Bouthier de la Tour Magali Toueille Françoise Vannier Suzanne Sommer Pascale Servant 《Molecular microbiology》2009,73(2):240-252
The nucleoid of radioresistant bacteria, including D . radiodurans , adopts a highly condensed structure that remains unaltered after exposure to high doses of irradiation. This structure may contribute to radioresistance by preventing the dispersion of DNA fragments generated by irradiation. In this report, we focused our study on the role of HU protein, a nucleoid-associated protein referred to as a histone-like protein, in the nucleoid compaction of D. radiodurans. We demonstrate, using a new system allowing conditional gene expression, that HU is essential for viability in D. radiodurans . Using a tagged HU protein and immunofluorescence microscopy, we show that HU protein localizes all over the nucleoid and that when HU is expressed from a thermosensitive plasmid, its progressive depletion at the non-permissive temperature generates decondensation of DNA before fractionation of the nucleoid into several entities and subsequent cell lysis. We also tested the effect of the absence of Dps, a protein also involved in nucleoid structure. In contrast to the drastic effect of HU depletion, no change in nucleoid morphology and cell viability was observed in dps mutants compared with the wild-type, reinforcing the major role of HU in nucleoid organization and DNA compaction in D. radiodurans . 相似文献
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Cloning and expression in Escherichia coli of a gene,hup, encoding the histone-like protein HU of Bifidobacterium longum 总被引:3,自引:0,他引:3
A genomic DNA library of Bifidobacterium longum ATCC15707 was transfected into an Escherichia coli strain deficient in both HU and IHF, the growth of which is cold-sensitive because of the deficiency in these proteins. Cold-resistant colonies were selected and the DNA was cloned and sequenced. A polypeptide consisted of 93 amino acids, a predicted molecular mass of 9983 Da with an isoelectric point of 10.35, was deduced from an orf in the middle of the DNA fragment. The amino acid sequence was highly similar to HU family proteins, and 26 aas of N terminal was identical to a histone-like protein, HBI, a HU family protein of B. longum. Incapabilities of Mu phage propagation in an E. coli mutant deficient in HU or IHF could be suppressed by DNA bearing this orf. These results showed that the orf is a gene hup encoding HBI, a histone-like protein HU of B. longum. 相似文献
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Several basic proteins, including the ubiquitous HU proteins, serve histone-like functions in prokaryotes. Significant sequence conservation exists between HU homologues; yet binding sites varying from 9 to 37 bp have been reported. TF1, an HU homologue with a 37 bp binding site that is encoded by the Bacillus subtilis bacteriophage SPO1, binds with nM affinity to DNA that contains 5-hydroxymethyluracil (hmU) in place of thymine and to T-containing DNA with loops. We evaluated the contribution of three conserved lysines to specifying the length of the binding site and show that Lys3 is critical for maintaining a long binding site in T-containing DNA: A mutant protein in which Lys3 is replaced with Gln(TF1-K3Q) is completely deficient in forming a stable complex. The affinity for 37 bp hmU-containing DNA is also reduced, from approximately 3 nM for wild-type TF1 to approximately 90 nM for TF1-K3Q. The decrease in affinity of TF1-K3Q for hmU-containing DNA > or = 25 bp suggests that Lys3 contacts DNA 8-9 bp distal to the sites of kinking. We propose that Lys3 forms an internal saltbridge to Asp26 in HU homologues characterized by shorter binding sites and that its surface exposure, and hence a longer binding site, may correlate with absence of this aspartate. 相似文献
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pAMβ1 resolvase has an atypical recombination site and requires a histone-like protein HU 总被引:1,自引:1,他引:0
The broad-host-range plasmid pAMβ1 from Gram-positive bacteria encodes a resolvase, designated Resβ, which shares homology with the proteins of the resolvase—invertase family. Here we report the purification and in vitro characterization of Resβ. This resolvase is particular in two aspects: it has an atypical binding site and requires a cofactor to promote resolution in vitro . Resβ binds to two regions within its resolution site res . One contains two inverted repeats (R1 and R2), the other contains only one repeat (R3). The cofactor required for resolution in vitro is present in crude extracts of both Bacillus subtilis and Escherichia coli and can be substituted by the E. coli histone-like protein HU. The possible mode of action of HU in the resolution process is discussed. 相似文献
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The HU protein is a small, basic, heat-stable DNA-binding protein that is well-conserved in prokaryotes and is associated with the bacterial nucleoid. In enterobacteria, including Escherichia coli, HU is a heterotypic dimer, HUalphabeta, composed of two closely related sub-units encoded by the hupA and hupB genes, respectively. HU was shown to participate in vitro in the initiation of DNA replication as an accessory factor to assist the action of DnaA protein in the unwinding of oriC DNA. To further elucidate the role of HU in the regulation of the DNA replication initiation process, we tested the synchrony phenotype in the absence of either one or both HU sub-units. The hupAB mutant exhibits an asynchronous initiation, the hupA mutant shows a similar reduced synchrony, whereas the hupB mutant shows a normal phenotype. Using a thermosensitive dnaA46 strain (dnaA46ts), an initiation mutant, we reveal a special role of HUbeta. The presence of a plasmid overproducing HUbeta in a dnaA46ts lacking HU (hupAB background) compensates for the thermosensitivity of this initiation mutant. Moreover, the overproduction of HUbeta confers to dnaA46ts a pattern of asynchrony similar to that of a dnaAcos, the intragenic suppressor of dnaA46ts. We show that the relative ratio of HUalpha versus HUbeta is greatly perturbed in dnaA46ts which accumulates little, if any, HUbeta. Therefore, the suppression of thermosensitivity in dnaA46hupAB by HUbeta may be caused by an unexpected absence of HUbeta in the dnaA46ts mutant. Visibly the HU composition is sensitive to the different states of DnaA, and may play a role during the regulation of the initiation process of the DNA replication by affecting subsequent events along the cell cycle. 相似文献
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The Escherichia coli histone-like protein HU affects DNA initiation, chromosome partitioning via MukB, and cell division via MinCDE.
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Escherichia coli hupA hupB double mutants, lacking both subunits (HU1 and HU2) of the histone-like protein HU, accumulate secondary mutations. In some genetic backgrounds, these include mutations in the minCDE operon, inactivating this system of septation control and resulting in the formation of minicells. In the course of the characterization of hupA hupB mutants, we observed that the simultaneous absence of the HU2 subunit and the MukB protein, implicated in chromosome partitioning, is lethal for the bacteria; the integrity of either HU or MukB thus seems to be essential for bacterial growth. The HU protein has been shown to be involved in DNA replication in vitro; we show here that its inactivation in the hupA hupB double mutant disturbs the synchrony of replication initiation in vivo, as evaluated by flow cytometry. Our results suggest that global nucleoid structure, determined in part by the histone-like protein HU, plays a role in DNA replication initiation, in proper chromosome partitioning directed by the MukFEB proteins, and in correct septum placement directed by the MinCDE proteins. 相似文献
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We report the purification of four proteins from Escherichia coli that stimulate or inhibit inter- and/or intramolecular recombination promoted by the yeast plasmid-encoded FLP protein. The proteins are identified as the ribosomal proteins S3 (27 kDa), L2 (26 kDa), S4 (24 kDa), and S5 (16 kDa), on the basis of N-terminal sequence analysis. The S3 protein is found to be identical to H protein, an E. coli histone-like protein that is related to histone H2A immunologically and by virtue of amino acid content. The H protein/S3 identity is based on co-migration on polyacrylamide gels, heat stability, amino acid analysis, and effects on FLP-promoted recombination. These results are relevant to current studies on the structure of the E. coli nucleoid. Since the H protein has previously been found associated with the E. coli nucleoid, the results indicate that either (a) some ribosomal proteins serve a dual function in E. coli, or, more likely, (b) ribosomal proteins can and are being mis-identified as nucleoid constituents. 相似文献
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Starved cultures of Escherichia coli are highly dynamic, undergoing frequent population shifts. The shifts result from the spread of mutants able to grow under conditions that impose growth arrest on the ancestral population. To analyze competitive interactions underlying this dynamic we measured the survival of a typical mutant and the wild type during such population shifts. Here we show that the survival advantage of the mutant at any given time during a takeover is inversely dependent on its frequency in the population, its growth adversely affects the survival of the wild type, and its ability to survive in stationary phase at fixation is lower than that of its ancestor. These mutants do not enter, or exit early, the nondividing stationary-phase state, cooperatively maintained by the wild type. Thus they end up overrepresented as compared to their initial frequency at the onset of the stationary phase, and subsequently they increase disproportionately their contribution in terms of progeny to the succeeding generation in the next growth cycle, which is a case of evolutionary cheating. If analyzed through the game theory framework, these results might be explained by the prisoner's dilemma type of conflict, which predicts that selfish defection is favored over cooperation. 相似文献
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Ramstein J Hervouet N Coste F Zelwer C Oberto J Castaing B 《Journal of molecular biology》2003,331(1):101-121
The Escherichia coli histone-like HU protein pool is composed of three dimeric forms: two homodimers, EcHUalpha(2) and EcHUbeta(2), and a heterodimer, EcHUalphabeta. The relative abundance of these dimeric forms varies during cell growth and in response to environmental changes, suggesting that each dimer plays different physiological roles. Here, differential scanning calorimetry and circular dichroism (CD) were used to study the thermal stability of the three E.coli HU dimers and show that each of them has its own thermodynamic signature. Unlike the other HU proteins studied so far, which melt through a single step (N(2)<-->2D), this present thermodynamic study shows that the three E.coli dimers melt according to a two-step mechanism (N(2)<-->I(2)<-->2D). The native dimer, N(2), melts partially into a dimeric intermediate, I(2), which in turn yields the unfolded monomers, D. In addition, the crystal structure of the EcHUalpha(2) dimer has been solved. Comparative thermodynamic and structural analysis between EcHUalpha(2) and the HU homodimer from Bacillus stearothermophilus suggests that the E.coli dimer is constituted by two subdomains of different energetic properties. The CD study indicates that the intermediate, I(2), corresponds to an HU dimer having partly lost its alpha-helices. The partially unfolded dimer I(2) is unable to complex with high-affinity, single-stranded break-containing DNA. These structural, thermodynamic and functional results suggest that the N(2)<-->I(2) equilibrium plays a central role in the physiology of E.coli HU. The I(2) molecular species seems to be the EcHUbeta(2) preferential conformation, possibly related to its role in the E.coli cold-shock adaptation. Besides, I(2) might be required in E.coli for the HU chain exchange, which allows the heterodimer formation from homodimers. 相似文献
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Preferential binding of E.coli histone-like protein HU alpha to negatively supercoiled DNA. 总被引:3,自引:0,他引:3
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Binding specificity of histone-like HU alpha protein to supercoiled DNA was examined by gel retardation assay and chemical probing with OsO4. The latter method was proved to be a unique means for detecting torsional tension restrained in supercoiled plasmid in the presence of HU alpha. It was shown that HU alpha protein has preferential affinity to negatively supercoiled DNA relative to relaxed, nicked and linearized DNAs. There were two modes for binding of HU alpha to the supercoiled DNA: one was the binding associated with topological changes in DNA and the other was relatively strong binding, probably specific to certain particular structures of DNA. It was suggested that HU in vivo interacts preferentially with the regions deformed under torsional stress or with the metabolically active regions along DNA. 相似文献