Regulation of Pkc1 Hyper-Phosphorylation by Genotoxic Stress

The cell wall integrity (CWI) signaling pathway is best known for its roles in cell wall biogenesis. However, it is also thought to participate in the response to genotoxic stress. The stress-activated protein kinase Mpk1 (Slt2, is activated by DNA damaging agents through an intracellular mechanism that does not involve the activation of upstream components of the CWI pathway. Additional observations suggest that protein kinase C (Pkc1), the top kinase in the CWI signaling cascade, also has a role in the response to genotoxic stress that is independent of its recognized function in the activation of Mpk1. Pkc1 undergoes hyper-phosphorylation specifically in response to genotoxic stress; we have found that this requires the DNA damage checkpoint kinases Mec1 (Mitosis Entry Checkpoint) and Tel1 (TELomere maintenance), but not their effector kinases. We demonstrate that the casein kinase 1 (CK1) ortholog, Hrr25 (HO and Radiation Repair), previously implicated in the DNA damage transcriptional response, associates with Pkc1 under conditions of genotoxic stress. We also found that the induced association of Hrr25 with Pkc1 requires Mec1 and Tel1, and that Hrr25 catalytic activity is required for Pkc1-hyperphosphorylation, thereby delineating a pathway from the checkpoint kinases to Pkc1. We used SILAC mass spectrometry to identify three residues within Pkc1 the phosphorylation of which was stimulated by genotoxic stress. We mutated these residues as well as a collection of 13 phosphorylation sites within the regulatory domain of Pkc1 that fit the consensus for CK1 sites. Mutation of the 13 Pkc1 phosphorylation sites blocked hyper-phosphorylation and diminished RNR3 (RiboNucleotide Reductase) basal expression and induction by genotoxic stress, suggesting that Pkc1 plays a role in the DNA damage transcriptional response.

Rapidly cycling stem cells regenerate the intestine independent of Lgr5high cells

The +4 cells in intestinal crypts are DNA damage-resistant and contribute to regeneration. However, their exact identity and the mechanism underlying +4 cell-mediated regeneration remain unclear. Using lineage tracing, we show that cells marked by an Msi1 reporter (Msi1+) are enriched at the +4 position in intestinal crypts and exhibit DNA damage resistance. Single-cell RNA sequencing reveals that the Msi1+ cells are heterogeneous with the majority being intestinal stem cells (ISCs). The DNA damage-resistant subpopulation of Msi1+ cells is characterized by low-to-negative Lgr5 expression and is more rapidly cycling than Lgr5high radio-sensitive crypt base columnar stem cells (CBCs); they enable fast repopulation of the intestinal epithelium independent of CBCs that are largely depleted after irradiation. Furthermore, relative to CBCs, Msi1+ cells preferentially produce Paneth cells during homeostasis and upon radiation repair. Together, we demonstrate that the DNA damage-resistant Msi1+ cells are rapidly cycling ISCs that maintain and regenerate the intestinal epithelium.

The Saccharomyces cerevisiae RAD6 Group Is Composed of an Error-Prone and Two Error-Free Postreplication Repair Pathways

The RAD6 postreplication repair and mutagenesis pathway is the only major radiation repair pathway yet to be extensively characterized. It has been previously speculated that the RAD6 pathway consists of two parallel subpathways, one error free and another error prone (mutagenic). Here we show that the RAD6 group genes can be exclusively divided into three rather than two independent subpathways represented by the RAD5, POL30, and REV3 genes; the REV3 pathway is largely mutagenic, whereas the RAD5 and the POL30 pathways are deemed error free. Mutants carrying characteristic mutations in each of the three subpathways are phenotypically indistinguishable from a single mutant such as rad18, which is defective in the entire RAD6 postreplication repair/tolerance pathway. Furthermore, the rad18 mutation is epistatic to all single or combined mutations in any of the above three subpathways. Our data also suggest that MMS2 and UBC13 play a key role in coordinating the response of the error-free subpathways; Mms2 and Ubc13 form a complex required for a novel polyubiquitin chain assembly, which probably serves as a signal transducer to promote both RAD5 and POL30 error-free postreplication repair pathways. The model established by this study will facilitate further research into the molecular mechanisms of postreplication repair and translesion DNA synthesis. In view of the high degree of sequence conservation of the RAD6 pathway genes among all eukaryotes, the model presented in this study may also apply to mammalian cells and predicts links to human diseases.