Chromatin was then eluted from your beads using elution buffer (1% SDS, 0.1 M NaHCO3) and the crosslinks reversed by incubation at 65C overnight. ORF3 are calculated by normalizing the ChIP -PCR signal with the input PCR signal. The value for UV untreated cells was set as 1.0. For each strain, data represent the mean 1 SD for three impartial experiments.(TIF) pone.0072090.s002.tif (164K) GUID:?4C7940C8-4F4B-49EA-B47C-443F42B1AC61 Physique S3: RNA polymerase II status during NER in different regions of the locus. B. Cells were irradiated with 100 J/m2 UV and incubated for different repair occasions as indicated. Chromatin was immunoprecipitated with 8WG16 antibody followed by quantitative PCR amplification using primers specific to ORF1, ORF2 and ORF3 of the locus in WT, WTRad26, H4 R45H and H4R45HRad26 cells. The values given for ORF1, ORF2 and ORF3 are calculated by normalizing the ChIP -PCR signal with the input PCR signal. The value for UV untreated cells was set as 1.0. For each strain, data represent the mean 1 SD for three impartial experiments.(TIF) pone.0072090.s003.tif (165K) GUID:?9F57CE00-D075-4A2A-AFA6-DCE50245C367 Abstract Transcription coupled nucleotide excision repair (TCR) is a major pathway responsible for removal of helix distorting DNA lesions from transcriptionally active regions of the genome. Rad26, a key factor of the TCR pathway, is known to play a role during early actions of TCR. Here, we show that Rad26-mediated TCR is not completely dependent on active transcription elongation in budding yeast. As per our results, gene is usually adversely affected in and D-Cycloserine however affects transcription of the constitutively expressed gene following UV-induced DNA damage repair. Chromatin immunoprecipitation (ChIP) D-Cycloserine analyses showed loss of RNAPII in the different ORF regions of constitutively expressed loci such as, and and loci of UV-irradiated gene, the cells were UV irradiated at 100 J/m2 and allowed to repair for different time periods, as indicated. Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed as described [27]. Mid-log phase yeast cells were treated with or without 100 D-Cycloserine J/m2 UV and allowed to repair for indicated time. Cells were then crosslinked with 1% formaldehyde and after suspension in lysis buffer (50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml pepstatin A) were disrupted using glass beads (425C600 m, Sigma), followed by sonication. Protein levels in the extract were estimated using the Bradford assay. Equal amounts of protein from each sample were used for immunoprecipitation with anti-RNA Polymerase II monoclonal antibody 8WG16 (Covance: MMS-126R). The reaction mixture was incubated overnight at 4C, and the immunocomplex precipitated using Protein A sepharose beads (50% slurry). The beads were consecutively washed with lysis buffer, wash buffer 1 (Lysis buffer made up of 500 mM NaCl), wash buffer D-Cycloserine 2 (10 mM TrisCHCl, pH 8, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA) and TE buffer and then treated with RNase A in TE at 37C for 30 min. Chromatin was then eluted from the beads using elution buffer (1% SDS, 0.1 M NaHCO3) and the crosslinks reversed by incubation at 65C overnight. Fragments representing specific ORF regions of locus were amplified from the immunoprecipitated DNA using sequence-specific primers. The forward and reverse primer sets used for ORF1, ORF2, and ORF3 of gene were and and and gene were: and and and respectively; for ORF1, ORF2, and ORF3 of gene were: and and and respectively. PCR products were resolved on 1.5% agarose gels. Experiments were repeated four occasions and the data is usually representative of the average of the different experimental sets. Results In trying to understand the role of Rad26 during TCR, we deleted from both wild type as well as the Sin mutant H4 R45H cells. Sin mutants are Swi/Snf Independent mutants and repair studies have shown that this Sin mutant H4 R45H is usually more resistant to UV irradiations and have faster nucleotide excision repair rate compared to wild type cells [26]. Transcriptome analysis revealed that under normal conditions 475 genes are up-regulated in H4 R45H cells compared to wild type. H4 R45H cells show high rates of transcription coupled NER in the constitutively active locus. deletion have distinctly adverse effect on the NER rate of both wild type and H4 R45H MULTI-CSF cells, the effect being more profound around the latter. Here we have tried to further our understanding around the role of Rad26 during UV-induced DNA damage response and transcription coupled NER. UV Sensitivity of.
