Rad26, the Transcription-Coupled Repair Factor in Yeast, Is Required for Removal of Stalled RNA Polymerase-II following UV Irradiation

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 steps of TCR. Here, we show that Rad26-mediated TCR is not absolutely dependent on active transcription elongation in budding yeast. As per our results, RAD26- deleted cells show enhanced UV sensitivity compared to wild type cells under conditions where transcription elongation is inhibited. The increased UV sensitivity observed in RAD26 -deleted cells, however, is not due to reduced expression of the major NER-responsive genes. Interestingly, transcription of the constitutively expressed RPB2 gene is adversely affected in RAD26- deleted cells during UV-induced DNA damage repair. In consonance, chromatin immunoprecipitation analysis showed that unlike in wild type, in RAD26 -deleted cells no significant reduction in RNA polymerase II occupancy occurs during nucleotide excision repair in the transcriptionally active loci like, RPB2 , PYK1 and RPL2B . These results collectively indicate that removal of RNAPII during DNA damage repair following UV irradiation is dependent on Rad26

Expression analyses of NER genes and <i>RPB2</i> gene.

<p>RT–PCR analysis was performed on total RNA isolated from WT and WTΔRad26 cells (<b>A</b>) following treatment without or with 100 J/m² UV radiation, using gene-specific primers, as described in Materials and Methods section; (<b>B</b>) of <i>RPB2</i> gene after 100 J/m² UV irradiation followed by repair incubation for different time periods. For each strain, data represent the mean ±1 SD for three independent experiments.</p

Sensitivity of cells to transcription elongation inhibitors with or without UV treatment.

<p>Cells of exponentially growing cultures were appropriately diluted and spread on SC plates supplemented with MPA (A) or 6-AU (B) of indicated concentrations without UV irradiation. Similarly grown cells were spread on SC plates supplemented with MPA (C) or 6-AU (D) and subjected to UV doses as indicated. Growth was monitored after 72 h. For each strain, data represent the mean ±1 SD for four independent experiments.</p

RNA polymerase II status during NER in different regions of the <i>RPB2</i> locus.

<p>ChIP analysis of RNA polymerase II status in different ORF regions of the <i>RPB2</i> locus as shown in (A), during NER. Cells were irradiated with 100 J/m² UV and incubated for different repair times as indicated. Chromatin was immunoprecipitated with 8WG16 antibody specific to RNA polymerase II, followed by quantitative PCR amplification using primers specific to ORF1 (B), ORF2 (C) and ORF3 (data not shown) of the <i>RPB2</i> locus in WT, H4 R45H, WTΔRad26 and H4R45HΔRad26 cells. The values given for ORF1 and ORF2 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 four independent experiments. Corresponding ChIP-PCR, input-PCR and no antibody control gel pictures are given below each strain.</p

UV sensitivity of cells.

<p>UV sensitivity of WT, H4 R45H, WTΔRad26 and H4 R45HΔrad26 cells. Colony forming ability following UV irradiation was monitored in exponentially growing cultures. Cells were appropriately diluted, spread on YPD plates, subjected to the UV doses shown and their survival monitored. For each strain, data represent the mean ±1 SD for four independent experiments.</p

Domain organization of Rad26 protein.

<p>Bioinformatics based studies indicated the Rad26 protein to be composed of primarily three distinct domains. The N-terminal aspartate/glutamate-rich acidic domain; a SNF2 domain having the ATP-binding helicase sub-domain consisting of the ATP-binding pocket and a signature DEGH box; and the C-terminal helicase domain.</p