Downregulation of the SL biosynthetic pathway in AR isolates.

<p>(A) Changes in the composition of major SL classes among the sequential isolates of <i>C. albicans</i> were assessed as described in methods. Green and black colour of gene in the pathway represents upregulation and no change, respectively. (B) Total SLs are represented as % of the total PGL + SE + SL mass spectral signal after normalization to internal standards and were determined as described in methods. (C) The fold change in expression levels of various SL biosynthetic pathway genes (relative amplification to ACT1) between TW1 (most susceptible to FLC) and TW17 (most resistant to FLC) were determined by RT-PCR. The gel picture is a representative of the RT-PCR analysis performed in replicates. Values in the histogram are means ± SD (n = 3 for all <i>Candida</i> strains). Asterisks “*” represents p<0.05. Lipid data taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039812#pone.0039812.s004" target="_blank">Sheet S1</a>, worksheet 4.</p

Measurement of mitochondrial membrane dysfunction among TW1 and TW17 isolates.

<p>For the flow cytometry analysis, cells were stained with 100 nM MitoTracker Red CMXRos (which fluoresces upon oxidation in respiring mitochondria). (A) Quadrant plots, (B) histograms and (C) bar graph of flow cytometry analysis between TW1 and TW17 are depicted (n = 4). (D) The red fluorescence of MitoTracker Red CMXRos was also visualized by confocal microscopy.</p

FLC exposure alters CW integrity in the sequential isolates of C. albicans.

<p>(A) Sequential isolates were tested with CW perturbing agents like Triton X-100 (upto 0.04%), 0.02% SDS, 16 µgml⁻¹ Congo Red. (B) Susceptibility to Calcofluor White (upto 50 µgml⁻¹) was tested. These spot tests were performed as described in methods. (C) Passive diffusion rate of PI was monitored by spectrofluorimeter. Briefly, the cells were pre-incubated with 3 µM PI for 45 min and then centrifuged. The supernatant was taken and the emission spectrum of PI was recorded for each strain. (D) The cells with PI were subjected to flow cytometry analysis to measure the amount of PI accumulated in each strain. The data is represented as the mean fluorescent intensity (n = 2).</p

Accumulation of odd chain-FA containing PGLs in the FLC resistant isolates.

<p>Total amount of odd chain FA-containing PGL was calculated by adding the normalized amounts of each odd chain FA containing PGL molecular species (namely 31-C, 33-C, 35-C and 37-C containing PGLs). The data is represented as % of total PGL + SL + SE mass spectral signal after normalization to internal standards. Values are mean of 3 independent analyses (n = 3). Asterisks “*” represents p < 0.05. Lipid data taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039812#pone.0039812.s004" target="_blank">Sheet S1</a> (worksheet 3), <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039812#pone.0039812.s006" target="_blank">Table S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039812#pone.0039812.s008" target="_blank">Table S3</a>.</p

Upregulation of the sterol biosynthetic pathway in AR isolates.

<p>(A) Changes in the composition of major sterols and its intermediates among the sequential isolates of <i>C. albicans</i> were analyzed as described in methods. Green and black colour of gene in the pathway (A) represents upregulation and no change, respectively. (B) Total SEs are represented as % of the total PGL+ SE + SL mass spectral signal after normalization to internal standards and were determined as described in methods. (C) The fold change in gene expression levels of various <i>ERG</i> genes (relative amplification to <i>ACT1</i>) between TW1 (most susceptible to FLC) and TW17 (most resistant to FLC) were determined by RT-PCR. The gel picture is a representative of the RT-PCR analysis performed in replicates. Values in the histogram are means ± SD (n = 3 for all <i>Candida</i> strains). Asterisks “*” represents p<0.05. Lipid data taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039812#pone.0039812.s004" target="_blank">Sheet S1</a>, worksheet 3 and 4.</p

FLC exposure leads to gradual development of a partially compromised CW and affects PG biosynthesis in several other clinical isolates of <i>C. albicans</i>.

<p>A. Several pairs of isogenic clinical isolates of <i>C. albicans</i> were tested with 0.02% SDS, a CW perturbing agent. The spot tests were performed as described in methods. B. PL analysis of various AS/AR clinically matched isogenic isolates by HP-TLC. Briefly, cells were grown overnight (to the exponential growth phase) in YEPD medium. Lipids were extracted as described earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039812#pone.0039812-Bligh1" target="_blank">[18]</a> and separated by thin-layer chromatography using chloroform: methanol: acetic acid (65:28:8) as the solvent system. HP-TLC analysis of each match pair and the corresponding values of each lipid class as mol % are also indicated.</p

Mammalian Base Excision Repair: Functional Partnership between PARP-1 and APE1 in AP-Site Repair

<div><p>The apurinic/apyrimidinic- (AP-) site in genomic DNA arises through spontaneous base loss and base removal by DNA glycosylases and is considered an abundant DNA lesion in mammalian cells. The base excision repair (BER) pathway repairs the AP-site lesion by excising and replacing the site with a normal nucleotide via template directed gap-filling DNA synthesis. The BER pathway is mediated by a specialized group of proteins, some of which can be found in multiprotein complexes in cultured mouse fibroblasts. Using a DNA polymerase (pol) β immunoaffinity-capture technique to isolate such a complex, we identified five tightly associated and abundant BER factors in the complex: PARP-1, XRCC1, DNA ligase III, PNKP, and Tdp1. AP endonuclease 1 (APE1), however, was not present. Nevertheless, the complex was capable of BER activity, since repair was initiated by PARP-1’s AP lyase strand incision activity. Addition of purified APE1 increased the BER activity of the pol β complex. Surprisingly, the pol β complex stimulated the strand incision activity of APE1. Our results suggested that PARP-1 was responsible for this effect, whereas other proteins in the complex had no effect on APE1 strand incision activity. Studies of purified PARP-1 and APE1 revealed that PARP-1 was able to stimulate APE1 strand incision activity. These results illustrate roles of PARP-1 in BER including a functional partnership with APE1.</p></div

Effect of PARP-1 on APE1-dependent BER.

<p>(A) A schematic representation of the DNA substrate containing the AP-site and the reaction scheme is shown. The BER reaction conditions and product analysis are described under Materials and Methods. (B) The BER reaction mixtures containing purified proteins XRCC1, PNKP, DNA ligase I and APE1 were supplemented either with PARP-1 (lanes 1–3) or dilution buffer (lanes 4–6). Repair was initiated by transferring the reaction mixtures to 37°C. Aliquots were withdrawn at 5, 10 and 20 min. The reaction products were analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124269#pone.0124269.g001" target="_blank">Fig 1</a>. The positions of the BER intermediate (unligated) and ligated BER products are indicated. (C) Quantification of the BER products was performed using ImageQuant software and data plotted as a function of incubation time (min). The plot demonstrates that BER product formation was linear during the 20 min incubation and that PARP-1 stimulated BER at least 2-fold as compared to the reaction without additional PARP-1.</p

Stimulation of APE1 activity by the pol β complex or purified PARP-1.

<p>A schematic representation of the DNA substrate containing the AP-site analogue THF is shown at the top. The reaction conditions and product analysis are described under Materials and Methods. (A) APE1 incision reactions were assembled on ice either with increasing amounts of pol β complex (A) or with increasing amounts of purified PARP-1 (B) The incision reaction was initiated by addition of 0.1 nM APE1 and transferring the reaction mixtures to 37°C for 10 min. The reaction products were analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124269#pone.0124269.g001" target="_blank">Fig 1</a>. The positions of the ³²P-labeled substrate and the product of APE1 strand incision are indicated. A representative phosphorimage of two repeats is illustrated. (C) Quantification of the APE1 product formed at the highest amount of pol β complex (3 μl) and the highest concentration of PARP-1 (50 nM) reveal an approximately 3-fold increase in APE1 activity as compared to that of APE1 alone. The mean of two repeats is illustrated.</p

Evidence for Abasic Site Sugar Phosphate-Mediated Cytotoxicity in Alkylating Agent Treated <em>Saccharomyces cerevisiae</em>

<div><p>To better understand alkylating agent-induced cytotoxicity and the base lesion DNA repair process in <em>Saccharomyces cerevisiae</em>, we replaced the <em>RAD27^FEN1</em> open reading frame (ORF) with the ORF of the bifunctional human repair enzyme DNA polymerase (Pol) β. The aim was to probe the effect of removal of the incised abasic site 5′-sugar phosphate group (i.e., 5′-deoxyribose phosphate or 5′-dRP) in protection against methyl methanesulfonate (MMS)-induced cytotoxicity. In <em>S. cerevisiae</em>, Rad27^Fen1 was suggested to protect against MMS-induced cytotoxicity by excising multinucleotide flaps generated during repair. However, we proposed that the repair intermediate with a blocked 5′-end, i.e., 5′-dRP group, is the actual cytotoxic lesion. In providing a 5′-dRP group removal function mediated by dRP lyase activity of Pol β, the effects of the 5′-dRP group were separated from those of the multinucleotide flap itself. Human Pol β was expressed in <em>S. cerevisiae</em>, and this partially rescued the MMS hypersensitivity observed with <em>rad27^fen1</em>-null cells. To explore this rescue effect, altered forms of Pol β with site-directed eliminations of either the 5′-dRP lyase or polymerase activity were expressed in <em>rad27^fen1</em>-null cells. The 5′-dRP lyase, but not the polymerase activity, conferred the resistance to MMS. These results suggest that after MMS exposure, the 5′-dRP group in the repair intermediate is cytotoxic and that Rad27^Fen1 protection against MMS in wild-type cells is due to elimination of the 5′-dRP group.</p> </div