The C-terminal zinc finger of UvrA does not bind DNA directly but regulates damage-specific DNA binding

In prokaryotic nucleotide excision repair, UvrA recognizes DNA perturbations and recruits UvrB for the recognition and processing steps in the reaction. One of the most remarkable aspects of UvrA is that it can recognize a wide range of DNA lesions that differ in chemistry and structure. However, how UvrA interacts with DNA is unknown. To examine the role that the UvrA C-terminal zinc finger domain plays in DNA binding, an eleven amino acid deletion was constructed (ZnG UvrA). Biochemical characterization of the ZnG UvrA protein was carried out using UvrABC DNA incision, DNA binding and ATPase assays. Although ZnG UvrA was able to bind dsDNA slightly better than wild-type UvrA, the ZnG UvrA mutant only supported 50-75% of wild type incision. Surprisingly, the ZnG UvrA mutant, while retaining its ability to bind dsDNA, did not support damage-specific binding. Furthermore, this mutant protein only provided 10% of wild-type Bca UvrA complementation for UV survival of an uvrA deletion strain. In addition, ZnG UvrA failed to stimulate the UvrB DNA damage-associated ATPase activity. Electrophoretic mobility shift analysis was used to monitor UvrB loading onto damaged DNA with wild-type UvrA or ZnG UvrA. The ZnG UvrA protein showed a 30-60% reduction in UvrB loading as compared with the amount of UvrB loaded by wild-type UvrA. These data demonstrate that the C-terminal zinc finger of UvrA is required for regulation of damage-specific DNA binding.     

Haemophilus influenzae UvrA: overexpression, purification, and in cell complementation

UvrA protein is a major component of ABC endonuclease complex involved in nucleotide excision repair (NER) mechanism. Although NER system is best characterized in Escherichia coli, not much information is available in Haemophilus influenzae. However, based on amino acid homology, uvrA ORF has been identified on H. influenzae genome [gene identification No. HI0249, Science 269 (1995) 496]. H. influenzae Rd uvrA ORF was cloned and overexpressed in E. coli. The expressed UvrA protein was purified using a two-step column chromatography protocol to a single band of expected molecular weight (104 kDa) and characterized for its ATPase and DNA binding activity. In addition, when H. influenzae uvrA was introduced in E. coli uvrA mutant strain AB1886, its UV resistance was restored to near wild type level.     

Identification and characterization of two <Emphasis Type="Italic">uvrA</Emphasis> genes of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pathovar citri

Two uvrA-like genes, designated uvrA1 and uvrA2, that may be involved in nucleotide excision repair in Xanthomonas axonopodis pv. citri (X. a. pv. citri) strain XW47 were characterized. The uvrA1 gene was found to be 2,964 bp in length capable of encoding a protein of 987 amino acids. The uvrA2 gene was determined to be 2,529 bp with a coding potential of 842 amino acids. These two proteins share 71 and 39% identity, respectively, in amino acid sequence with the UvrA protein of Escherichia coli. Analyses of the deduced amino acid sequence revealed that UvrA1 and UvrA2 have structures characteristic of UvrA proteins, including the Walker A and Walker B motifs, zinc finger DNA binding domains, and helix-turn-helix motif with a polyglycine hinge region. The uvrA1 or uvrA2 mutant, constructed by gene replacement, was more sensitive to DNA-damaging agents methylmethane sulfonate (MMS), mitomycin C (MMC), or ultraviolet (UV) than the wild type. The uvrA1 mutant was four orders of magnitude more sensitive to UV irradiation and two orders of magnitude more sensitive to MMS than the uvrA2 mutant. The uvrA1uvrA2 double mutant was one order of magnitude more sensitive to MMS, MMC, or UV than the uvrA1 single mutant. These results suggest that UvrA1 plays a more important role than UvrA2 in DNA repair in X. a. pv. citri. Both uvrA1 and uvrA2 genes were found to be constitutively expressed in the wild type and lexA1 or lexA2 mutant of X. a. pv. citri, and treatment of these cells with sublethal dose of MMC did not alter the expression of these two genes. Results of electrophoresis mobility shift assays revealed that LexA1 or LexA2 does not bind to either the uvrA1 or the uvrA2 promoter. These results suggest that uvrA expression in X. a. pv. citri is not regulated by the SOS response system.     

The Streptomyces peucetius drrC gene encodes a UvrA-like protein involved in daunorubicin resistance and production.

The drrC gene, cloned from the daunorubicin (DNR)- and doxorubicin-producing strain of Streptomyces peucetius ATCC 29050, encodes a 764-amino-acid protein with a strong sequence similarity to the Escherichia coli and Micrococcus luteus UvrA proteins involved in excision repair of DNA. Expression of drrC was correlated with the timing of DNR production in the growth medium tested and was not dependent on the presence of DNR. Since introduction of drrC into Streptomyces lividans imparted a DNR resistance phenotype, this gene is believed to be a DNR resistance gene. The drrC gene could be disrupted in the non-DNR-producing S. peucetius dnrJ mutant but not in the wild-type strain, and the resulting dnrJ drrC double mutant was significantly more sensitive to DNR in efficiency-of-plating experiments. Expression of drrC in an E. coli uvrA strain conferred significant DNR resistance to this highly DNR-sensitive mutant. However, the DrrC protein did not complement the uvrA mutation to protect the mutant from the lethal effects of UV or mitomycin even though it enhanced the UV resistance of a uvrA+ strain. We speculate that the DrrC protein mediates a novel type of DNR resistance, possibly different from the mechanism of DNR resistance governed by the S. peucetius drrAB genes, which are believed to encode a DNR antiporter.     

Degradation of intracellular protein in Salmonella typhimurium peptidase mutants

Multiply peptidase-deficient mutant strains of Salmonella typhimurium fail to carry out normal protein degradation during starvation for a carbon source. In these mutants, the extent of protein breakdown during starvation is about fourfold less than in the wild type. The products of protein breakdown in the mutant are mainly small, trichloroacetic acid-soluble peptides, not free amino acids as in the wild type. The carbon-starved mutant strain produces only about one thirtieth as much free amino acid from protein as the wild type. As a result, protein synthesis during starvation is reduced in the mutant compared to the wild type and the mutant strain shows a greatly prolonged lag phase after a nutritional shift-down.     

Controlled degradation by ClpXP protease tunes the levels of the excision repair protein UvrA to the extent of DNA damage

UV irradiation damages DNA and activates expression of genes encoding proteins helpful for survival under DNA stress. These proteins are often deleterious in the absence of DNA damage. Here, we investigate mechanisms used to regulate the levels of DNA-repair proteins during recovery by studying control of the nucleotide excision repair (NER) protein UvrA. We show that UvrA is induced after UV irradiation and reaches maximum levels between ∼20 and 120 min post UV. During post-UV recovery, UvrA levels decrease principally as a result of ClpXP-dependent protein degradation. The rate of UvrA degradation depends on the amount of unrepaired pyrimidine dimers present; this degradation rate is initially slow shortly after UV, but increases as damage is repaired. This increase in UvrA degradation as repair progresses is also influenced by protein–protein interactions. Genetic and in vitro experiments support the conclusion that UvrA–UvrB interactions antagonize degradation. In contrast, Mfd appears to act as an enhancer of UvrA turnover. Thus, our results reveal that a complex network of interactions contribute to tuning the level of UvrA in the cell in response to the extent of DNA damage and nicely mirror findings with excision repair proteins from eukaryotes, which are controlled by proteolysis in a similar manner.     

Effect of adenosine 5''-triphosphate-dependent deoxyribonuclease deficiency on properties and transformation of Haemophilus influenzae strains.

A transformation-deficient strain of Haemophilus influenzae, lacking adenosine 5'-triphosphate-dependent deoxyribonuclease activity, was isolated by selection for sensitivity to mitomycin. The mutant, designated JK57, possibily showed a moderate sensitivity to ultraviolet (UV) irradiation and treatment with methyl methane sulfonate. Contrary to the wild type, the mutant degraded chromosomal deoxyribonucleic acid (DNA) to some extent. However, after UV irradiation to the mutant degraded considerably less DNA than the wild type and the TD24 mutant of H. influenzae, the latter being equivalent to a recA mutant of Escherichia coli. A TD2457 double mutant, constructed by transferring the TD24 mutation into the JK57 strain, was as sensitive to deleterious agents and as deficient in transformation as the TD24 single mutant; in the double mutant, however, after UV irradiation chromosomal DNA was degraded to the same extent as in the JK57 mutant. The number of transformants per unit of radioactive donor DNA taken up by JK57 recipient cells was approximately 10-fold smaller than in the wild type. Presynaptically, the fate of donor DNA in the adenosine 5'-triphosphate-dependent deoxyribonuclease-deficient mutants was not different from that in the wild type. In contrast to TD24 and the TD2457 double mutant, in the JK57 mutant, recombinant-type activities (molecules carrying both the donor and recipient markers) were formed almost as well as in the wild type. After integration into the JK57 recipient genome, the rate of replication of the donor marker was equal to that of the recipient marker during a number of generations, which suggests that the donor DNA is normally integrated into the JK57 chromosome. It is suggested that transformed JK57 cells pass with a high frequency into a type of cells that can replicate their chromosomes many times but have lost the ability to form visible colonies after plating.     

Amplified UvrA protein can ameliorate the ultraviolet sensitivity of an Escherichia coli recA mutant.

When a recA strain of Escherichia coli was transformed with the multicopy plasmid pSF11 carrying the uvrA gene of E. coli, its extreme ultraviolet (UV) sensitivity was decreased. The sensitivity of the lexA1 (Ind(-)) strain to UV was also decreased by pSF11. The recA cells expressing Neurospora crassa UV damage endonuclease (UVDE), encoding UV-endonuclease, show UV resistance. On the other hand, only partial amelioration of UV sensitivity of the recA strain was observed in the presence of the plasmid pNP10 carrying the uvrB gene. Host cell reactivation of UV-irradiated lambda phage in recA cells with pSF11 was as efficient as that in wild-type cells. Using an antibody to detect cyclobutane pyrimidine dimers, we found that UV-irradiated recA cells removed dimers from their DNA more rapidly if they carried pSF11 than if they carried a vacant control plasmid. Using anti-UvrA antibody, we observed that the expression level of UvrA protein was about 20-fold higher in the recA strain with pSF11 than in the recA strain without pSF11. Our results were consistent with the idea that constitutive level of UvrA protein in the recA cells results in constitutive levels of active UvrABC nuclease which is not enough to operate full nucleotide excision repair (NER), thus leading to extreme UV sensitivity.     

Interaction of nystatin with nystatin-resistantCandida tropicalis

Nystatin-resistant yeast Candida tropicalis was obtained after UV illumination and plating on nystatin-containing media. The mutant contained no ergosterol in the plasma membrane but bound nystatin to a degree similar to that of the wild strain (1.2 vs. 1.5 nmol per mg dry solid). Respiration of the mutant on glucose was reduced by 36% in the presence of 25 microM nystatin. This corresponded to a 25-43% decrease of the uptake of monosaccharides. Transport of amino acids was reduced by nystatin in the mutant by 44-86%, as compared with a 84-95% reduction in the wild strain. The intracellular ATP content was reduced by nystatin equally in the wild strain and in the mutant (by 43 and 47%). Nystatin appears to affect specifically membrane transport processes of nonelectrolytes while both the H+-extruding ATPase and the membrane potential are unaffected.     

The beta -hairpin motif of UvrB is essential for DNA binding, damage processing, and UvrC-mediated incisions.

UvrB plays a major role in recognition and processing of DNA lesions during nucleotide excision repair. The crystal structure of UvrB revealed a similar fold as found in monomeric DNA helicases. Homology modeling suggested that the beta-hairpin motif of UvrB might be involved in DNA binding (Theis, K., Chen, P. J., Skorvaga, M., Van Houten, B., and Kisker, C. (1999) EMBO J. 18, 6899-6907). To determine a role of the beta-hairpin of Bacillus caldotenax UvrB, we have constructed a deletion mutant, Deltabetah UvrB, which lacks residues Gln-97-Asp-112 of the beta-hairpin. Deltabetah UvrB does not form a stable UvrB-DNA pre-incision complex and is inactive in UvrABC-mediated incision. However, Deltabetah UvrB is able to bind to UvrA and form a complex with UvrA and damaged DNA, competing with wild type UvrB. In addition, Deltabetah UvrB shows wild type-like ATPase activity in complex with UvrA that is stimulated by damaged DNA. In contrast to wild type UvrB, the ATPase activity of mutant UvrB does not lead to a destabilization of the damaged duplex. These results indicate that the conserved beta-hairpin motif is a major factor in DNA binding.     

Micrococcus luteus homolog of theEscherichia coli uvrA gene: Identification of a mutation in the UV-sensitive mutant DB7

Summary Restriction fragments ofMicrococcus luteus DNA containing the gene affected by a mutation in the UV-sensitive mutant DB7 were cloned both from the wild type and from the mutant in anEscherichia coli host-vector system. The wild-type fragment was able to reverse the multiple sensitivity of the mutant to UV, mitomycin C, and 4-nitroquinoline 1-oxide by a one-step transformation. Determination of the nucleotide sequences revealed a potential open reading frame coding for a protein of 992 (tentative) amino acid residues, within which the DB7 mutation was identified as a CG-to-TA transition causing a translation termination. The putative product of the open reading frame shares an extensive amino acid sequence homology with theE. coli UvrA protein comprising 940 residues. The homology extends over the greater part of both polypeptides except for two extra sequences of 31 and 24 amino acid residues located at the amino-terminal and in the interior, respectively, of theM. luteus protein. In the homologous region, 56.7% and 16.7% of the 933 pairs of the aligned amino acids were accounted for by conserved residues and conservative substitutions, respectively. These results indicate that the gene defined by the mutation in DB7 represents a homolog of theE. coli uvrA gene. Hence, it has to be concluded that DB7, known for its deficiency in UV endonuclease (pyrimidine dimer DNA glycosylase/apurinic-apyrimidinic endonuclease) activity, is a double mutant which is also defective in an enzyme complex similar to theE. coli UvrABC excinuclease. Dedicated to the memory of Shunzo Okubo (1930–1978), who considered the possibility of a double-mutant status for the mutant DB7 most seriously     

Triple point mutation Asp10-->His, Asn101-->Asp, Arg148-->Ser in T4 phage lysozyme leads to the molten globule.

The triple amino acid replacement (Asp10-->His, Asn101-->Asp, Arg148-->Ser) in T4 phage lysozyme was carried out by site-directed mutagenesis. At acid pH (2.7) the mutant is in a conformational state with the properties of the molten globule: (i) the mutant protein molecule is essentially compact; (ii) its CD spectrum in the near UV region is drastically reduced in intensity as compared with the wild type protein spectrum; (iii) the CD spectrum in the far UV region indicates the presence of pronounced secondary structure in the mutant; (iv) unlike the wild type protein the mutant protein can bind the hydrophobic fluorescent probe, ANS.     

meoA is the structural gene for outer membrane protein c of Escherichia coli K12

Summary The isolation and characterization of two mutants of Escherichia coli K12 with an altered outer membrane protein c is described. The first mutant, strain CE1151, was isolated as a bacteriophage Mel resistant strain which contains normal levels of protein c. Mutant cells adsorbed the phage with a strongly decreased rate. Complexes of purified nonheat modified wild type protein c and wild type lipopolysaccharide inactivated phage Me1, indicating that these components are required for receptor activity for phage Me1. When wild type protein c was replaced by protein c of strain CE1151, the receptorcomplex was far less active, showing that protein c of strain CE1151 is altered. The second mutant produces a protein c with a decreased electrophoretic mobility, designated as protein c^*. An altered apparent molecular weight was also observed for one or more fragments obtained after fragmentation of the mutant protein with cyanogen bromide, trypsin and chymotrypsin. Alteration of protein c was not accompanied by a detectable alteration in protein b or its fragments. Both mutations are located at minute 48 of the Escherichia coli K12 linkage map. The results strongly suggest that meoA is the structural gene for protein c.     

Excision-repair capacity in Streptococcus pneumoniae: cloning and expression of a uvr-like gene

Although deficient in photoreactivation and some SOS-like functions, Streptococcus pneumoniae has the capacity to carry out excision repair when exposed to UV light. The repair ability and sensitivity to UV irradiation or treatment with chemical agents in the wild type and a UV-sensitive mutant strain indicate that UV-induced pyrimidine dimers might be repaired in pneumococcus by a system similar to the uvr-dependent system in Escherichia coli. A gene complementing the mutation conferring UV sensitivity of the mutant strain has been cloned. The coding region directs the synthesis of a polypeptide with a molecular weight of 78 kDa. The relationship with uvr-like protein in E. coli is discussed.     

The effect of reducing light-harvesting pigment on marine microalgal productivity

The productivity was evaluated of a strain of Chlamydomonas perigranulata isolated from the RedSea. A mutant with small light-harvesting pigments(LHC-1) was obtained by UV mutagenesis. Thechlorophylls content of the wild type was twice ashigh as that of LHC-1, and the initial slope of thephotosynthesis-irradiance curve was higher in the wildtype. However, the maximum photosynthetic activity ona per cell basis was almost the same. It isconcluded that LHC-1 is a mutant with lesslight-harvesting pigment (LHP) than the wild type. Aspreviously reported, the mutant with lower LHP contenthas a higher productivity in a continuous culturesystem, so we compared the productivity of the wildtype and the mutant. The maximum productivity of LHC-1was 1.5 times higher than that of the wild type. Itis suggested that the technique of reducing thecontent of light-harvesting pigment should be madeavailable for other organisms.     

Involvement of the Reserve Material Poly-β-Hydroxybutyrate in Azospirillum brasilense Stress Endurance and Root Colonization

When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-β-hydroxybutyrate (PHB). Azospirillum brasilense strain Sp7 and a phbC (PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth. The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested. The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain. The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type. However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited. The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains. In soil, the two strains colonized roots to the same extent. It appears that synthesis and utilization of PHB as a carbon and energy source by A. brasilense under stress conditions favor establishment of this bacterium and its survival in competitive environments. However, in A. brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested.     

耐辐射球菌基因DRB0099缺失突变株的构建及逆境分析

耐辐射球菌(Deinococcus radiodurans R1)有着极强的辐射抗性.研究其抗辐射的机理对于处理放射性废料有着潜在的应用价值.在耐辐射球菌的基因组中,许多序列的功能未知.其中DRB0099尤为引人注意.将DRB0099缺失突变构建该基因的突变株.对野生型和突变体进行比较后发现,在正常生长条件下的前期阶段(0～16 h),突变体生长速度比野生型慢.16 h以后,野生型逐渐进入稳定生长期.这时,突变株的生长速度高于野生型.但是,野生型的浓度一直高于突变株.表明在DRB0099被删除后,耐辐射球菌的生长可能受到了阻滞.在紫外线照射的条件下,尽管野生型随着照射剂量的增加,存活率越来越低,但是要比突变体高许多.野生型具有比突变体更强的修复DNA双链断裂的能力.DRB0099可能直接参与了对DNA的修复.突变体对H2O2的敏感程度高于野生型,表明野生型耐辐射球菌在对抗活性氧保护其蛋白质、DNA或者DNA修复方面具有比突变体更强的功能.在低浓度H2O2处理条件下,尽管野生型和突变体的存活率都出现下降趋势,但二者的差值并不大.随着H2O2剂量的增加,二者的差值越来越大.表明随着活性氧浓度的增加,蛋白质和DNA损伤的数量增加,失去DRB0099基因功能的突变体比野生型更容易受到损伤.在紫外线照射处理或者H2O2处理条件下,DRB0099能够保护蛋白质和DNA.     

Both ATPase sites of Escherichia coli UvrA have functional roles in nucleotide excision repair.

The roles of the two tandemly arranged putative ATP binding sites of Escherichia coli UvrA in UvrABC endonuclease-mediated excision repair were analyzed by site-directed mutagenesis and biochemical characterization of the representative mutant proteins. Evidence is presented that UvrA has two functional ATPase sites which coincide with the putative ATP binding motifs predicted from its amino acid sequence. The individual ATPase sites can independently hydrolyze ATP. The C-terminal ATPase site has a higher affinity for ATP than the N-terminal site. The invariable lysine residues at the ends of the glycine-rich loops of the consensus Walker type "A" motifs are indispensable for ATP hydrolysis. However, the mutations at these lysine residues do not significantly affect ATP binding. UvrA, with bound ATP, forms the most favored conformation for DNA binding. The initial binding of UvrA to DNA is chiefly at the undamaged sites. In contrast to the wild type UvrA, the ATPase site mutants bind equally to damaged and undamaged sites. Dissociation of tightly bound nucleoprotein complexes from the undamaged sites requires hydrolysis of ATP by the C-terminal ATPase site of UvrA. Thus, both ATP binding and hydrolysis are required for the damage recognition step enabling UvrA to discriminate between damaged and undamaged sites on DNA.