Rpd3L Contributes to the DNA Damage Sensitivity ofSaccharomyces cerevisiaeCheckpoint Mutants

Abstract

DNA replication forks that are stalled by DNA damage activate an S-phase checkpoint that prevents irreversible fork arrest and cell death. The increased cell death caused by DNA damage in budding yeast cells lacking the Rad53 checkpoint protein kinase is partially suppressed by deletion of the EXO1 gene. Using a whole-genome sequencing approach, we identified two additional genes, RXT2 and RPH1, whose mutation can also partially suppress this DNA damage sensitivity. We provide evidence that RXT2 and RPH1 act in a common pathway, which is distinct from the EXO1 pathway. Analysis of additional mutants indicates that suppression works through the loss of the Rpd3L histone deacetylase complex. Our results suggest that the loss or absence of histone acetylation, perhaps at stalled forks, may contribute to cell death in the absence of a functional checkpoint.

Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation

“…Indeed, MRC1 8D rad53Δ sml1Δ cells showed some improvement in survival, compared to rad53Δ sml1Δ cells when chronically exposed to low concentrations of either HU (2 mM) or MMS (0.006%), though they did not promote additional survival to higher concentrations (8 mM HU and 0.01% MMS) (Fig 6D). Similar results were obtained with 2 freshly germinated spores of each genotype (Fig S4 ) arguing that the suppression seen was not a result of suppressor mutations, which accumulate readily in rad53Δ sml1Δ cells (Gómez-González et al, 2019). Figure 6E shows that MRC1 8D also promoted increased survival to acute exposure to higher concentration of MMS (0.02%) relative to MRC1 wild type cells (Fig 6E).…”

Rad53 limits CMG helicase uncoupling from DNA synthesis at replication forks

“…Our observation that Rad53 further restricts DNA unwinding by the CMG provides a molecular mechanism for the previously observed checkpoint-dependent control of replisome progression 10 , 11 , 13 , 21 , 23 . Restriction of replisome progression by Rad53 may limit the generation of ssDNA to prevent replication catastrophe from RPA exhaustion and protect forks from nuclease attack 57 , 58 . CMG inhibition by Rad53 may also help maintain the replisome near the site of polymerase stalling to facilitate the recoupling of CMG to leading strand synthesis.…”

Rpd3L and Hda1 histone deacetylases facilitate repair of broken forks by promoting sister chromatid cohesion

“…Interestingly, cohesins accumulate at stalled replication forks and seem required for efficient fork progression after DNA damage 53,54 . However, it is worthy to note that the action of Rpd3L was reported to be toxic in the S-phase checkpoint mutants 55 . Our results imply that although histone deacetylation can have deleterious consequences at stalled forks, it can also be beneficial for repair and fork progression and suggest that histone acetylation levels must be finely regulated to accomplish proper genome duplication.…”