Rpb9-deficient cells are defective in DNA damage response and require histone H3 acetylation for survival

Rpb9 is a non-essential subunit of RNA polymerase II that is involved in DNA transcription and repair. In budding yeast, deletion of RPB9 causes several phenotypes such as slow growth and temperature sensitivity. We found that simultaneous mutation of multiple N-terminal lysines within histone H3 was lethal in rpb9Δ cells. Our results indicate that hypoacetylation of H3 leads to inefficient repair of DNA double-strand breaks, while activation of the DNA damage checkpoint regulators γH2A and Rad53 is suppressed in Rpb9-deficient cells. Combination of H3 hypoacetylation with the loss of Rpb9 leads to genomic instability, aberrant segregation of chromosomes in mitosis, and eventually to cell death. These results indicate that H3 acetylation becomes essential for efficient DNA repair and cell survival if a DNA damage checkpoint is defective.

. Depletion of Rpb9 is lethal in H3 K9,14,23R cells. Anchor-away strains with Rpb9 depletion system along with wild-type strain (AKY796) and rpb9Δ mutant (AKY1037) were spotted in 10-fold dilutions onto YPD plates containing DMSO (control) or 1µg/ml rapamycin (for Rpb9 depletion). Plates were photographed after 2 days of incubation. Genetic backgrounds of the strains are summarized in the table. FRB tag (fused to C-terminus of Rpb9 protein) and RPL13A-2xFKBP12 are essential components of the anchor-away system; tor1-1 mutation is required for bypass from rapamycin-induced cell cycle arrest.  Figure S2. 10-fold serial dilutions of Rpb9 anchor-away strains along with RAD53 and rad53Δ strains expressing wt H3 or the H3 K9,14,23R mutant were spotted onto synthetic compleyte (SC) plates containing DMSO (+Rpb9) or rapamycin (-Rpb9). Cells were treated with ionizing radiation (50-150 Gy), or plated on media containing 30 µg/ml camptothecin (CPT). Strains AKY1162 and AKY1190 express rpb9-FRB protein that can be removed from cell nucleus by anchor-away technique. rad52Δ strain (AKY1518) is sensitive to various DNA damaging agents and it was used as a positive control for DNA damage induction. Plates were photographed after 3 days of growth at 30°C. Figure S3. Western blot analysis of H2A (A and B) and Rad53 (C and D) phosphorylation in response to MMS treatment in Rpb9-depleted cells. Rpb9 anchor-away strains with wt or K9,14,23R mutant H3 were incubated with DMSO (+Rpb9) or rapamycin (-Rpb9) for 6 hours before 0.01% MMS was added to the cells and samples were taken at indicated time-points. Western blot analysis of H3 (E and F) is shown as a loading control. A positive sample from unrelated experiment was loaded as marker (lane "M" on blots). Glucose + rapamycin (or DMSO) (3h, 6h, 20h) 1.

Experimental scheme:
HO endonuclease Glucose Galactose

HO cut site
Yeast cells express HO endonuclease under the control of galactose-inducible promoter. In glucose-or raffinose-containing medium, expression of HO endonuclease is repressed. When the cells are shifted to galactose-containing medium, expression of HO is induced and the nuclease makes a DSB at its recognition site in the MAT locus.
The formed DNA break is repaired primarily by homologous recombination (HR) using the gene cassettes in HMLα or HMRa loci as donors. As long as the HO endonuclease is expressed, the locus is continuously cut and repaired. During the course of the experiment, the intactness of the MAT locus is detected by PCR. When cells are grown in galactose-containing medium, the majority of cells contain a DSB in HO recognition site and amplification of the MAT locus by PCR is strongly reduced. When HO expression is shut-down in glucose-containing medium, the locus is repaired and can be detected by PCR. Depending on the donor sequence used for DSB repair, the lenght of the PCR product can be either 1.0 kb (MATa) or 1.1 kb (MATα).
1. Cells are pre-grown in raffinose-containing medium. Expression of HO endonuclease is OFF.
2. Expression of HO nuclease is induced in galactose-containing medium for 1.5 hours to induce DSBs in the MAT locus.
3. Rapamycin (or DMSO) is added to growth medium to induce depletion of Rpb9 protein from the cell nucleus. Cells are grown for further 1.5 hours. Expression of HO endonuclease is ON. 4. Expression of HO endonuclease is shut-off by transferring the cells into glucose-containing medium. DNA samples are collected at different time-points to estimate the efficiency of MAT locus repair in the presence or absence of Rpb9 protein in the cell nucleus (DMSO and rapamycin samples, respectively). Figure S4. Overview of the DSB repair assay. In all strains the galactose-inducible HO endonuclease is integrated into ade3 locus and Rpb9 protein can be removed from cell nucleus by the anchor-away technique. HO cuts its recognition site in the MAT locus in chromosome III. The site is repaired mainly by homologous recombination using the HMLα or HMRa loci in the same chromosome as donor sequences. Location of the chromosome III centromere relative to the other loci is indicated as a blue circle. Red arrow above the MAT locus indicates the HO cut site and black bars under the MAT locus indicate the region amplified by PCR to estimate the intactness of the locus. Figure S5. (A and B) Agarose gel electrophoresis analysis of HO cutting and repair. PCR products were obtained from cells before HO induction (Raf), 3 hours after HO induction (Gal) and 3, 6 or 20 hours after repression of HO (Glc). PCR product of the VPS13 locus was used as an internal control. Thermo Scientific GeneRuler 1 kb DNA ladder (M) was used. (B) Different PCR cycles (19,21,26) were tested to make sure that the PCR product formation was in linear range.