Notably, in a KO, S263A had no additional reduction in NHEJ repair efficiency, but S263E recovered the KO NHEJ defect (Figure ?(Figure6D).6D). NHEJ efficiency, and similarly, PRKACB was found to phosphorylate XLF (a Nej1 human homolog) at S263, a corresponding residue of the yeast Nej1 S298. Together, our results uncover a new and conserved mechanism for Tpk1 and PRKACB in phosphorylating Nej1 (or XLF), which is critically required for NHEJ repair. Graphical Abstract Open in a separate windows Graphical Abstract Cellular model of yeast Tpk1 role with Nej1 and PRKACBwith XLF on NHEJ, as well as the loss of tpk1 in DNA resection and MMEJ. INTRODUCTION DNA double-stranded breaks (DSBs) are the most severe forms of DNA damage, causing genomic instability and chromosomal rearrangements (1), leading to serious human diseases including cancer and immunodeficiency response (2,3). In response to DSBs, eukaryotic cells recruit three repair pathways: homologous recombination (HR), non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) to rescue genomic instability. NHEJ is the main DSB repair pathway in mammalian cells, and highly conserved in eukaryotes ranging from the budding yeast, genes, and one regulatory subunit, encoded by gene (15,16). Components of the PKA catalytic subunit trimeric complex (Tpk1-3) exhibit varied substrate specificities (17), indicating the potential for distinct functional profiles. A yeast proteome chip screening approach (17) has uncovered Nej1 as a phosphorylation substrate of Tpk1, but not Tpk2 SJG-136 or Tpk3, implying a likely SJG-136 role for Tpk1 in DNA repair. Our recent work has shown deletion impaired NHEJ (18), and that it is epistatic with a yeast uncharacterized gene, (HU resistance 1) in NHEJ, though the molecular mechanism of this connection in DNA repair remains unclear (19). While this and other evidence suggests the role of yeast SJG-136 PKA in DNA damage checkpoint pathways (13), and Tpk1 in NHEJ (18,19), how PKA subunits impact these processes, and whether there is a conserved role between yeast and human in DSB repair pathway, warrants further investigation. In the present study, we show that deletion of and (or the checkpoint kinase, kinase activity dependent on Nej1 serine residue at S298, a phosphosite of Dun1, suggesting a crosstalk between the two DNA damage checkpoint kinases (Tpk1, Dun1) through SJG-136 shared phosphorylation of the Nej1 S298. As well, the repair defect of deletion can be recovered by mutation of Nej1 S298E (phosphomimetic), but not Nej1 S298A (non-phosphorylatable), consistent with the phosphorylation of S298 being a requisite for Tpk1s role in NHEJ repair. As in yeast, PRKACB, a human homolog of Tpk1, showed comparable NHEJ defect in DSB repair by phosphorylating the Nej1 human homolog, XLF at S263, suggesting a conserved NHEJ role for Tpk1 in eukaryotes. Overall, our findings suggest that Nej1 phosphorylation by Tpk1 is usually intrinsic to NHEJ break repair and resolution, and spotlight a conserved model for NHEJ regulation. MATERIALS AND METHODS Besides the procedures described below, site-directed mutagenesis, kinase and phosphorylation-induced mobility shift, sensitivity to DNA damage, clonogenic survival, cell proliferation, phosphoproteomics/affinity purification coupled with mass spectrometry (MS), immunoblotting, purification of SJG-136 yeast Tpk1 and Nej1 recombinant proteins, generation of stable CRISPR gene knockouts (KOs), immunofluorescence staining and quantification of DNA damage foci, and alkaline comet assay, among others are detailed in Supplementary Methods. Mutant strains or CRISPR gene KOs, cell cultures and plasmids All yeast gene deletions (or mammalian gene KOs), plasmids, primers, and antibodies used are listed in Supplementary Table S1. Yeast Mouse monoclonal to CD54.CT12 reacts withCD54, the 90 kDa intercellular adhesion molecule-1 (ICAM-1). CD54 is expressed at high levels on activated endothelial cells and at moderate levels on activated T lymphocytes, activated B lymphocytes and monocytes. ATL, and some solid tumor cells, also express CD54 rather strongly. CD54 is inducible on epithelial, fibroblastic and endothelial cells and is enhanced by cytokines such as TNF, IL-1 and IFN-g. CD54 acts as a receptor for Rhinovirus or RBCs infected with malarial parasite. CD11a/CD18 or CD11b/CD18 bind to CD54, resulting in an immune reaction and subsequent inflammation FLAG or HA-tagged fusion proteins and mutant strains were created in BY4741 or JKM139 background by lithium acetate transformation (20). Deletions were confirmed by PCR and/or DNA sequencing across the deletion site. Yeast strains were produced in YPD (1% yeast extract, 2% bacto-peptone, 2% glucose) at 30C, unless otherwise noted. Human osteosarcoma U2OS and HEK293T cells were cultured in Dulbecco’s altered Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin mix at 37C and 5% CO2. Stable human gene KOs were created using a CRISPR-Cas9 system with LentiCRISPR-v2 blasticidin or puromycin, following established procedure (21). NHEJ.