Preferential repair of bulky DNA adducts from the transcribed genes via nucleotide excision repair is well characterized in mammalian cells. However, definitive evidence is lacking for similar repair of oxidized bases, the major endogenous DNA lesions. Here we show that the oxidized base-specific human DNA glycosylase NEIL2 associates with RNA polymerase II and the transcriptional regulator heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), both in vitro and in cells. NEIL2 immunocomplexes from cell extracts preferentially repaired the mutagenic cytosine oxidation product 5-hydroxyuracil in the transcribed strand. In a reconstituted system, we also observed NEIL2-initiated transcription-dependent base excision repair of 5-hydroxyuracil in the transcribed strand, with hnRNP-U playing a critical role. Chromatin immunoprecipitation/reimmunoprecipitation studies showed association of NEIL2, RNA polymerase II, and hnRNP-U on transcribed but not on transcriptionally silent genes. Furthermore, NEIL2-depleted cells accumulated more DNA damage in active than in silent genes. These results strongly support the preferential role of NEIL2 in repairing oxidized bases in the transcribed genes of mammalian cells.
We have cloned and overexpressed multidrug transporter CaMdr1p as a green fluorescent proteintagged protein to show its capability to extrude drug substrates. The drug extrusion was sensitive to pH and energy inhibitors and displayed selective substrate specificity. CaMdr1p has a unique and conserved antiporter motif, also called motif C [G(X 6 )G(X 3 )GP(X 2 )GP(X 2 )G], in its transmembrane segment 5 (TMS 5). Alanine scanning of all the amino acids of the TMS 5 by site-directed mutagenesis highlighted the importance of the motif, as well as that of other residues of TMS 5, in drug transport. The mutant variants of TMS 5 were placed in four different categories. The first category had four residues, G244, G251, G255, and G259, which are part of the conserved motif C, and their substitution with alanine resulted in increased sensitivity to drugs and displayed impaired efflux of drugs. Interestingly, first category mutants, when replaced with leucine, resulted in more dramatic loss of drug resistance and efflux. Notwithstanding the location in the core motif, the second category included residues which are part of the motif, such as P260, and those which were not part of the motif, such as L245, W248, P256, and F262, whose substitution with alanine resulted in a severe loss of drug resistance and efflux. The third category included G263, which is a part of motif C, but unlike other conserved glycines, its replacement with alanine or leucine showed no change in the phenotype. The replacement of the remaining 11 residues of the fourth category did not result in any change. The putative helical wheel projection showed clustering of functionally critical residues to one side and thus suggests an asymmetric nature of TMS 5.
The repair of reactive oxygen species-induced base lesions and single strand breaks (SSBs) in the nuclear genome via the base excision (BER) and SSB repair (SSBR) pathways, respectively, is well characterize, and important for maintaining genomic integrity. However, the role of mitochondrial (mt) BER and SSBR proteins in mt genome maintenance is not completely clear. Here we show the presence of the oxidized base-specific DNA glycosylase Nei-like 2 (NEIL2) and the DNA end-processing enzyme polynucleotide kinase 3 -phosphatase (PNKP) in purified human mitochondrial extracts (MEs). Confocal microscopy revealed co-localization of PNKP and NEIL2 with the mitochondrion-specific protein cytochrome c oxidase subunit 2 (MT-CO2). Further, chromatin immunoprecipitation analysis showed association of NEIL2 and PNKP with the mitochondrial genes MT-CO2 and MT-CO3 (cytochrome c oxidase subunit 3); importantly, both enzymes also associated with the mitochondrion-specific DNA polymerase ␥. In cell association of NEIL2 and PNKP with polymerase ␥ was further confirmed by proximity ligation assays. PNKP-depleted ME showed a significant decrease in both BER and SSBR activities, and PNKP was found to be the major 3 -phosphatase in human ME. Furthermore, individual depletion of NEIL2 and PNKP in human HEK293 cells caused increased levels of oxidized bases and SSBs in the mt genome, respectively. Taken together, these studies demonstrate the critical role of NEIL2 and PNKP in maintenance of the mammalian mitochondrial genome.Reactive oxygen species-induced genomic damage includes a plethora of oxidized bases, apurinic/apyrimidinic (AP) 3 sites, and DNA single strand breaks (SSBs) that are often mutagenic and are etiologically linked to various pathophysiologies, including sporadic cancer, and a multitude of age-related degenerative diseases (1, 2). All of these DNA base lesions are primarily repaired by the evolutionarily conserved base excision repair (BER) pathway in both the nucleus and mitochondria (3). BER for oxidized bases is initiated by the recognition and cleavage of the base lesion from DNA by a DNA glycosylase; the resulting AP site is then cleaved by the intrinsic lyase activity of the glycosylase. In mammalian cells, five oxidized base-specific DNA glycosylases have been identified; they belong to two families based on their reaction mechanisms and structural conservation of their catalytic motifs. OGG1 and NTH1 of the Nth family remove base lesions only from duplex DNA and have -elimination activity, generating 3Ј-deoxyribose phosphate and 5Ј-phosphate (5Ј-P) groups at the resulting SSB (4). In contrast, the recently discovered NEILs 1-3 of the Nei family are active with duplex as well as single-stranded DNA (occurring transiently during DNA replication and transcription). Our recent studies now show that NEIL2 preferentially repairs oxidative damage from the transcribed genes, and NEIL1 primarily associates with the replication-associated proteins, thus indicating its involvement in repair during DNA replication (5, 6...
In this study, we investigated the role of cellular iron status in hyphae and biofilm formation in Candida albicans. Iron deprivation by a chelator, bathophenanthrolene disulfonic acid, promoted hyphal development even in nonhyphal-inducing media without affecting growth of C. albicans cells. Iron-acquisition defective mutants, Deltaftr1 and Deltaccc2, also showed hyphal formation, which was prevented by iron supplementation. Notably, most of the tested morphological mutants Deltacph1, Deltaefh1 and Deltatpk1 continued to form hyphae under iron-deprived conditions, except the Deltaefg1 null mutant, which showed a complete block in hyphae formation. The role of EFG1 in filamentation under iron-deprived conditions was further confirmed by Northern analysis, which showed a considerable upregulation of the EFG1 transcript. Of notable importance, all the morphological mutants including Deltaefg1 mutant possessed enhanced membrane fluidity under iron-deprived conditions; however, this did not appear to contribute to hyphal development. Interestingly, iron deprivation did not affect the ability of C. albicans to form biofilms on the catheter surface and led to no gross defects in azole resistance phenotype of these biofilms of C. albicans cells. Our study, for the first time, establishes a link between cellular iron, Efg1p and hyphal development of C. albicans cells that is independent of biofilm formation.
Candida drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, confers multidrug resistance in immunocompromised and debilitated patients. A member of the ATP-binding cassette (ABC) superfamily of membrane transporters, Cdr1p contains two nucleotide binding/utilization sites (NBDs) and two transmembrane domains (TMDs). We had earlier characterized Cdr1p by its overexpression as a GFP-tagged fusion protein that elicits oligomycin-sensitive ATPase activity and is linked to drug extrusion. However, it is essential to have highly purified Cdr1p to understand the detailed molecular basis of structure and functions of this protein. In this study, we have developed a two-step purification protocol using stably overexpressed His-tagged Cdr1p in Saccharomyces cerevisiae. Purified Cdr1p exhibited divalent cation-dependent ATPase activity [approximately 1.2 micromol (mg of protein)(-)(1) min(-)(1)] with an apparent K(M) in the range of 1.8 to 2.1 mM and V(max) between 1.0 and 1.4 micromol (mg of protein)(-)(1) min(-)(1). Unlike its close homologue human P-gp/MDR1, purified Cdr1p only moderately displayed drug stimulated ATPase activity. By exploiting intrinsic fluorescence intensity of purified Cdr1p, which contains 24 tryptophan residues, we could monitor defined conformational changes upon substrate drug and ATP binding. It is observed that ATP binding to Cdr1p (K(d) = approximately 1.7 mM) is not a prerequisite for drug binding, and both the mechanisms of drug as well as ATP binding, which induce specific conformational changes, occur independent of each other. Our study for the first time provides a catalytically active purified ABC transporter from a fungal pathogen, which is amenable to fluorescence measurements and thus would be useful in understanding the molecular basis of antifungal transport.
Steroids are known to induce pleiotropic drug resistance states in hemiascomycetes, with tremendous potential consequences for human fungal infections. Our analysis of gene expression in Saccharomyces cerevisiae and Candida albicans cells subjected to three different concentrations of progesterone revealed that their pleiotropic drug resistance (PDR) networks were strikingly sensitive to steroids. In S. cerevisiae, 20 of the Pdr1p/Pdr3p target genes, including PDR3 itself, were rapidly induced by progesterone, which mimics the effects of PDR1 gain-of-function alleles. This unique property allowed us to decipher the respective roles of Pdr1p and Pdr3p in PDR induction and to define functional modules among their target genes. Although the expression profiles of the major PDR transporters encoding genes ScPDR5 and CaCDR1 were similar, the S. cerevisiae global PDR response to progesterone was only partly conserved in C. albicans. In particular, the role of Tac1p, the main C. albicans PDR regulator, in the progesterone response was apparently restricted to five genes. These results suggest that the C. albicans and S. cerevisiae PDR networks, although sharing a conserved core regarding the regulation of membrane properties, have different structures and properties. Additionally, our data indicate that other as yet undiscovered regulators may second Tac1p in the C. albicans drug response.
The objectives of the study were to monitor and assess the road traffic noise in its spatial-temporal aspect in an urban area. The paper discusses the observations, results and their interpretation based on the study. Noise recordings from site, collected from April 2006 to March 2006, were used for statistical analysis and generation of various noise indices. Noise maps were also created for impact analysis and formulation of Noise Risk Zones. Mean Ldn value ranged between 55.1 and 87.3 dB (A). Day time Leq level ranged between 51.2 and 89.0 dB (A), where it ranged between 43.5 and 81.9 dB (A) during night. The study reveals that present noise level in all the locations exceeds the limit prescribed by CPCB. Based on the finding it can be said that the population in this industrial town are exposed to significantly high noise level, which is caused mostly due to road traffic.
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