Specific DNA binding and regulation of its own expression by the AidB protein in Escherichia coli

Upon exposure to alkylating agents, Escherichia coli increases expression of aidB along with three genes (ada, alkA, and alkB) that encode DNA repair proteins. While the biological roles of the Ada, AlkA, and AlkB proteins have been defined, despite many efforts, the molecular functions of AidB remain largely unknown. In this study, we focused on the biological role of the AidB protein, and we demonstrated that AidB shows preferential binding to a DNA region that includes the upstream element of its own promoter, PaidB. The physiological significance of this specific interaction was investigated by in vivo gene expression assays, demonstrating that AidB can repress its own synthesis during normal cell growth. We also showed that the domain architecture of AidB is related to the different functions of the protein: the N-terminal region, comprising the first 439 amino acids (AidB "I-III"), possesses FAD-dependent dehydrogenase activity, while its C-terminal domain, corresponding to residues 440 to 541 (AidB "IV"), displays DNA binding activity and can negatively regulate the expression of its own gene in vivo. Our results define a novel role in gene regulation for the AidB protein and underline its multifunctional nature.     

The AidB component of the Escherichia coli adaptive response to alkylating agents is a flavin-containing, DNA-binding protein

Upon exposure to alkylating agents, Escherichia coli increases expression of aidB along with three genes (ada, alkA, and alkB) that encode DNA repair proteins. In order to begin to identify the role of AidB in the cell, the protein was purified to homogeneity, shown to possess stoichiometric amounts of flavin adenine dinucleotide (FAD), and confirmed to have low levels of isovaleryl-coenzyme A (CoA) dehydrogenase activity. A homology model of an AidB homodimer was constructed based on the structure of a four-domain acyl-CoA oxidase. The predicted structure revealed a positively charged groove connecting the two active sites and a second canyon of positive charges in the C-terminal domain, both of which could potentially bind DNA. Three approaches were used to confirm that AidB binds to double-stranded DNA. On the basis of its ability to bind DNA and its possession of a redox-active flavin, AidB is predicted to catalyze the direct repair of alkylated DNA.     

Flavin-induced oligomerization in Escherichia coli adaptive response protein AidB

The process known as "adaptive response" allows Escherichia coli to respond to small doses of DNA-methylating agents by upregulating the expression of four proteins. While the role of three of these proteins in mitigating DNA damage is well understood, the function of AidB is less clear. Although AidB is a flavoprotein, no catalytic role has been established for the bound cofactor. Here we investigate the possibility that flavin plays a structural role in the assembly of the AidB tetramer. We report the generation and biophysical characterization of deflavinated AidB and of an AidB mutant that has greatly reduced affinity for flavin adenine dinucleotide (FAD). Using fluorescence quenching and analytical ultracentrifugation, we find that apo AidB has a high affinity for FAD, as indicated by an apparent dissociation constant of 402.1 ± 35.1 nM, and that binding of substoichiometric amounts of FAD triggers a transition in the AidB oligomeric state. In particular, deflavinated AidB is dimeric, whereas the addition of FAD yields a tetramer. We further investigate the dimerization and tetramerization interfaces of AidB by determining a 2.8 ? resolution crystal structure in space group P3(2) that contains three intact tetramers in the asymmetric unit. Taken together, our findings provide strong evidence that FAD plays a structural role in the formation of tetrameric AidB.     

Fission yeast Drp1 is an essential protein required for recovery from DNA damage and chromosome segregation

DNA double strand breaks (DSBs) are the most critical types of DNA damage that can leads to chromosomal aberrations, genomic instability and cancer. Several genetic disorders such as Xeroderma pigmentosum are linked with defects in DNA repair. Human Rint1, a TIP1 domain containing protein is involved in membrane trafficking but its role in DNA damage response is elusive. In this study we characterized the role of Drp1 (damage responsive protein 1), a Rint1 family protein during DNA damage response in fission yeast. We identified that Drp1 is an essential protein and indispensable for survival and growth. Using in vitro random mutagenesis approach we isolated a temperature sensitive mutant allele of drp1 gene (drp1-654) that exhibits sensitivity to DNA damaging agents, in particular to alkylation damage and UV associated DNA damage. The drp1-654 mutant cells are also sensitive to double strand break inducing agent bleomycin. Genetic interaction studies identified that Rad50 and Drp1 act in the same pathway during DNA damage response and the physical interaction of Drp1 with Rad50 was unaffected in drp1-654 mutant at permissive as well as non permissive temperature. Furthermore Drp1 was found to be required for the recovery from MMS induced DNA damage. We also demonstrated that the Drp1 protein localized to nucleus and was required to maintain the chromosome stability.     

Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS

Background

Brucella abortus is the etiological agent of a worldwide zoonosis called brucellosis. This alpha-proteobacterium is dividing asymmetrically, and PdhS, an essential histidine kinase, was reported to be an old pole marker.

Results

We were interested to identify functions that could be recruited to bacterial poles. The Brucella ORFeome, a collection of cloned predicted coding sequences, was placed in fusion with yellow fluorescent protein (YFP) coding sequence and screened for polar localizations in B. abortus. We report that AidB-YFP was systematically localized to the new poles and at constrictions sites in B. abortus, either in culture or inside infected HeLa cells or RAW264.7 macrophages. AidB is an acyl-CoA dehydrogenase (ACAD) homolog, similar to E. coli AidB, an enzyme putatively involved in destroying alkylating agents. Accordingly, a B. abortus aidB mutant is more sensitive than the wild-type strain to the lethality induced by methanesulphonic acid ethyl ester (EMS). The exposure to EMS led to a very low frequency of constriction events, suggesting that cell cycle is blocked during alkylation damage. The localization of AidB-YFP at the new poles and at constriction sites seems to be specific for this ACAD homolog since two other ACAD homologs fused to YFP did not show specific localization. The overexpression of aidB, but not the two other ACAD coding sequences, leads to multiple morphological defects.

Conclusions

Data reported here suggest that AidB is a marker of new poles and constriction sites, that could be considered as sites of preparation of new poles in the sibling cells originating from cell division. The possible role of AidB in the generation or the function of new poles needs further investigation.     

Repair of O6-methyl-guanine residues in DNA takes place by a similar mechanism in extracts from hela cells,human liver,and rat liver

Extracts from HeLa S₃ cells, human liver, and rat liver were found to contain an activity that transfers the methyl group from O⁶-methyl-guanine residues in DNA to a cysteine residue of an acceptor protein. The molecular weights of the acceptor proteins in HeLA cells and human liver are 24,000 ± 1,000 and 23,000 ± 1,000. respectively. Assuming that each acceptor molecule is used only once, the average number of acceptor molecules in HeLa cells was calculated to be about 50,000. The extracts also contained 3-methyl-adenine-DNA glycosylase activity and 7-methyl-guanine-DNA glycosylase activity, although the latter activity was not detected in extracts from human liver in our assay system. Thus, the three major alkylation products resulting from the effect of methylating agents, such as N-methyl-N-nitroso urea, can all be repaired in animal cells. Pretreatment of HeLa cells with N-methyl-N′-nitro-N-nitrosoguanidinc (0.1 μg/ml) strongly reduced the capacity of HeLa cell extracts to repair O⁶-methyl-guanine residues, while the activity of three DNA-N-glycosylases was essentially unaltered. This inactivation was not caused by a direct methylation of the enzyme by the carcinogen. The results demonstrate that the mechanism of repair of O⁶-methyl-guaninc residues, in DNA is strikingly similar in E coli and animal cells, including humans.     

Repair of endogenous DNA base lesions modulate lifespan in mice

The accumulation of DNA damage is thought to contribute to the physiological decay associated with the aging process. Here, we report the results of a large-scale study examining longevity in various mouse models defective in the repair of DNA alkylation damage, or defective in the DNA damage response. We find that the repair of spontaneous DNA damage by alkyladenine DNA glycosylase (Aag/Mpg)-initiated base excision repair and O⁶-methylguanine DNA methyltransferase (Mgmt)-mediated direct reversal contributes to maximum life span in the laboratory mouse. We also uncovered important genetic interactions between Aag, which excises a wide variety of damaged DNA bases, and the DNA damage sensor and signaling protein, Atm. We show that Atm plays a role in mediating survival in the face of both spontaneous and induced DNA damage, and that Aag deficiency not only promotes overall survival, but also alters the tumor spectrum in Atm^−/− mice. Further, the reversal of spontaneous alkylation damage by Mgmt interacts with the DNA mismatch repair pathway to modulate survival and tumor spectrum. Since these aging studies were performed without treatment with DNA damaging agents, our results indicate that the DNA damage that is generated endogenously accumulates with age, and that DNA alkylation repair proteins play a role in influencing longevity.     

Kin3 protein,a NIMA-related kinase of Saccharomyces cerevisiae,is involved in DNA adduct damage response

Kin3 is a nonessential serine/threonine protein kinase of the budding yeast Saccharomyces cerevisiae with unknown cellular role. It is an ortholog of the Aspergillus nidulans protein kinase NIMA (Never-In Mitosis, gene A), which is involved in the regulation of G2/M phase progression, DNA damage response and mitosis. The aim of this study was to determine whether Kin3 is required for proper checkpoint activation and DNA repair. Here we show that KIN3 gene deficient cells present sensitivity and fail to arrest properly at G2/M-phase checkpoint in response to the DNA damage inducing agents MMS, cisplatin, doxorubicin and nitrogen mustard, suggesting that Kin3 can be involved in DNA strand breaks recognition or signaling. In addition, there is an increase in KIN3 gene expression in response to the mutagenic treatment, which was confirmed by the increase of Kin3 protein. We also showed that co-treatment with caffeine induces a slight increase in the susceptibility to genotoxic agents in kin3 cells and abolishes KIN3 gene expression in wild-type strain, suggesting that Kin3p can play a role in Tel1/Mec1-dependent pathway activation induced after genotoxic stress. These data provide the first evidence of the involvement of S. cerevisiae Kin3 in the DNA damage response.     

DNA-methylation by nitrosocimetidine and N-methyl-N′-nitro-N-nitrosoguanidine in the intact rat

The ability of the nitroso derivative of the drug cimetidine to interact with cellular macromolecules in the intact rat was investigated. Radiolabelled nitrosocimetidine (NC) was shown to methylate DNA in a variety of tissues in the rat after oral administration. Radioactivity was also detected in the RNA and protein extracted from these same tissues. Methylation of DNA by the parent compound, cimetidine, was not detected in any of the tissues studied. For comparison, the DNA methylation produced by the carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) dosed orally was measured. DNA alkylation by MNNG was found to be approx. 2–36 times greater than that produced by NC, varying with the tissues studied. The highest yield of DNA alkylation was found in the stomach for MNNG and the small intestine for nitrosocimetidine suggesting pharmacokinetic differences.     

Some chemical aspects of dose-response relationships in alkylation mutagenesis

Alkylation of DNA can lead to induction of potentially miscoding groups (promutagenic) or potentially template-inactivating groups (lethal). The proportions of these are found to vary with the chemical nature of the alkylating agent. Agents of low Swain and Scott s factor (or those tending to Ingold's S_Ni type) react relatively more extensively at O-atom sites in DNA, and yield relatively more of the miscoding O₆-alkylguanine residues. Phosphotriester formation is also relatively more extensive with S_Ni agents.Inactivation of DNA can result from depurinations, strand breakage, and cross-linkage.Both promutagenic and lethal lesions are subject to repair; 3 principal enzymatic systems appear to exist; one for excision and repair of cross-links or aralkyl groups resembles the uvr system; others for repair of single-strand breaks parallel repair of X-ray-induced breaks (exr, rec systems); another, less well defined at present, recognizes certain methylated bases, and depurinated sites (probably Goldthwait's endonuclease II).These factors can be shown to influence dose-response in alkylation mutagenesis. This, broadly, can be classified as linear with the promutagenic group-inducing or directly miscoding agents, and is independent of cytotoxicity; whereas with other agents non-linear response parallels the occurrence of “shouldered” survival curves, and reflects mutation induction by “repairs errors”.Additionally, alkylation of cellular constituents other than DNA, e.g. repair enzymes, may influence dose response, and will again depend on chemical reactivity of the agent.     

Tsw gene-based resistance is triggered by a functional RNA silencing suppressor protein of the Tomato spotted wilt virus

As a result of contradictory reports, the avirulence (Avr) determinant that triggers Tsw gene-based resistance in Capsicum annuum against the Tomato spotted wilt virus (TSWV) is still unresolved. Here, the N and NSs genes of resistance-inducing (RI) and resistance-breaking (RB) isolates were cloned and transiently expressed in resistant Capsicum plants to determine the identity of the Avr protein. It was shown that the NSs^RI protein triggered a hypersensitive response (HR) in Tsw-containing Capsicum plants, but not in susceptible Capsicum, whereas no HR was discerned after expression of the N^RI/RB protein, or when NSs^RB was expressed. Although NSs^RI was able to suppress the silencing of a functional green fluorescence protein (GFP) construct during Agrobacterium tumefaciens transient assays on Nicotiana benthamiana, NSs^RB had lost this capacity. The observation that RB isolates suppressed local GFP silencing during an infection indicated a recovery of RNA silencing suppressor activity for the NSs protein or the presence of another RNA interference (RNAi) suppressor. The role of NSs as RNA silencing suppressor and Avr determinant is discussed in the light of a putative interplay between RNAi and the natural Tsw resistance gene.     

Derepression of single-stranded DNA-binding protein genes on plasmids derepressed for conjugation, and complementation of an E. coli ssb- mutation by these genes

Summary Plasmid single-stranded DNA-binding protein genes complement the E. coli ssb-1 mutation, and partially restore capacity for DNA synthesis, DNA repair (direct role as well as role in SOS induction) and general recombination. Plasmid mutants derepressed for fertility derived from R1, R64 and R222 show a higher level of complementation compared to the parental repressed plasmids. Derepressed mutants of R222 synthesize more RNA which hybridizes with the ssb gene of the F factor than does the original R222 plasmid. This indicates that plasmid ssb genes are regulated coordinately with fertility genes.     

Interaction of flavodoxin with cyanobacterial thylakoids

Flavodoxin from the cyanobacterium Anabaena PCC 7119 has been shown to mediate, under illumination, the transfer of electrons from the thylakoidal membranes that were isolated from the same organism, to both the enzyme ferredoxin-NADP⁺ reductase and cytochrome c. Chemical cross-linking of ferredoxin or flavodoxin to the photosynthetic membranes provides a preparation that is active in cytochrome c photoreduction without the addition of external protein carrier. NADP⁺ photoreduction, albeit diminished, was observed only after addition of exogenous electron carrier protein. Immunoblotting analysis of the chemical adduct reveals that flavodoxin binds to a 10 kDa polypeptide subunit in the cyanobacterial Photosystem I which appears to act as its physiological partner in the electron transfer process.Abbreviations Fd ferredoxin - Fld flavodoxin - cyt c cytochrome c - EDC 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide - PS I Photosystem I     

Similar degree of methyl methanesulphonate-induced DNA damage in two clones ofTradescantia differing in mutagenic sensitivity to alkylating agents

In two clones ofTradescantia (4430 and 02) differing in the sensitivity to the mutagenic action of alkylating agents, equimolar doses of [¹⁴C] methyl methanesulphonate (MMS) elicited a similar degree of protein, RNA and DNA alkylation and a similar amount of DNA-7-methylguanine and DNA-3-methyladenine in cells of inflorescence. Moreover, in the same clones and tissues the same doses of nonlabelled MMS produced a similar amount of DNA single strand breaks and/or alkali labile sites as measured in alkaline sucrose gradients. None of the DNA lesions followed is therefore decisive for explanation of the different mutagenic sensitivity ofTradescantia clones.     

Effects of herbicide 2,4-dinitrophenol on mitosis,DNA, RNA and protein synthesis inNigella sativa L.

Effect of 2,4-dinitrophenol (DNP) was studied onNigella sativa to note the changes in mitosis, DNA, RNA and protein synthesis. The chemical affected division frequency considerably and chromosomal abnormalities like sticky bridge, fragmentation, micronucleietc. were recorded. By using precursors of nucleic acid and protein synthesis, it was found that DNP also inhibited DNA, RNA and protein synthesis. The decrease in division frequency can be correlated with the DNA synthesis.