The DNA-dependent protein kinase catalytic subunit (DNA-PKcs; encoded by PRKDC) functions in DNA non-homologous end-joining (NHEJ), the major DNA double strand break (DSB) rejoining pathway. NHEJ also functions during lymphocyte development, joining V(D)J recombination intermediates during antigen receptor gene assembly. Here, we describe a patient with compound heterozygous mutations in PRKDC, low DNA-PKcs expression, barely detectable DNA-PK kinase activity, and impaired DSB repair. In a heterologous expression system, we found that one of the PRKDC mutations inactivated DNA-PKcs, while the other resulted in dramatically diminished but detectable residual function. The patient suffered SCID with reduced or absent T and B cells, as predicted from PRKDC-deficient animal models. Unexpectedly, the patient was also dysmorphic; showed severe growth failure, microcephaly, and seizures; and had profound, globally impaired neurological function. MRI scans revealed microcephaly-associated cortical and hippocampal dysplasia and progressive atrophy over 2 years of life. These neurological features were markedly more severe than those observed in patients with deficiencies in other NHEJ proteins. Although loss of DNA-PKcs in mice, dogs, and horses was previously shown not to impair neuronal development, our findings demonstrate a stringent requirement for DNA-PKcs during human neuronal development and suggest that high DNA-PK protein expression is required to sustain efficient pre-and postnatal neurogenesis. Introduction DNA nonhomologous end-joining (NHEJ) represents the major DNA double strand break (DSB) repair process in mammalian cells (1, 2). Thus, cells, mice, and patients defective in NHEJ proteins show markedly reduced DSB repair and radiosensitivity. NHEJ also functions during immune development due to its requisite role in V(D)J recombination (3). V(D)J recombination mediates immunoglobulin and T cell receptor gene assembly from variable (V), diversity (D), and joining (J) gene segments. Recombination is initiated by the RAG1/2 endonuclease, which introduces DSBs at recombination signal sequences (RSSs). The DNA ends, including the sequences encoding the antigen receptors (termed coding ends), have hairpinned termini; the RSSs are blunt ended. Rejoining of these recombination intermediates requires NHEJ proteins. Consequently, NHEJ defects in animals and humans causes SCID with reduced or absent T and B cells.6 core NHEJ components assemble as 2 complexes (1). The Ku70/80 heterodimer is the DNA double-stranded (ds) end recognition protein, binding DNA ds ends with high affinity. Ku recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs; encoded by PRKDC), generating the DNA-PK holoenzyme. The DNA-PK complex prevents unbridled exonuclease digestion of DNA ends, enhances appropriate end-processing, and recruits the ligation complex, which encompasses XRCC4,
PARPi efficacy in BRCA1-deficient cells depends on 53BP1 and shieldin, which have been proposed to limit single-stranded DNA at DSBs by blocking resection and/or through CST/Polα/primase-mediated fill-in. We show that—like 53BP1/shieldin and CST/Polα—primase promotes radial chromosome formation in PARPi-treated BRCA1-deficient cells and demonstrate shieldin/CST/Polα/primase-dependent incorporation of BrdU at DSBs. In the absence of 53BP1 or shieldin, radial formation in BRCA1-deficient cells was restored by tethering of CST near DSBs, arguing that in this context shieldin acts primarily by recruiting CST. Furthermore, a SHLD1 mutant defective in CST binding (SHLD1Δ) was non-functional in BRCA1-deficient cells and its function was restored upon reconnecting SHLD1Δ to CST. Interestingly, at dysfunctional telomeres and DNA breaks in class switch recombination where CST has been implicated, SHLD1Δ was fully functional, perhaps because these DNA ends carry CST recognition sites that afford SHLD1-independent binding of CST. The data establish that in BRCA1-deficient cells, CST/Polα/primase is the major effector of shieldin-dependent DSB processing.
Cdc7-Dbf4 kinase or DDK (Dbf4-dependent kinase) is required to initiate DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells and is an emerging chemotherapeutic target since DDK inhibition causes apoptosis of diverse cancer cell types but not of normal cells. PHA-767491 and XL413 are among a number of potent DDK inhibitors with low nanomolar IC50 values against the purified kinase. Although XL413 is highly selective for DDK, its activity has not been extensively characterized on cell lines. We measured anti-proliferative and apoptotic effects of XL413 on a panel of tumor cell lines compared to PHA-767491, whose activity is well characterized. Both compounds were effective biochemical DDK inhibitors but surprisingly, their activities in cell lines were highly divergent. Unlike PHA-767491, XL413 had significant anti-proliferative activity against only one of the ten cell lines tested. Since XL413 did not effectively inhibit DDK in multiple cell lines, this compound likely has limited bioavailability. To identify potential leads for additional DDK inhibitors, we also tested the cross-reactivity of ∼400 known kinase inhibitors against DDK using a DDK thermal stability shift assay (TSA). We identified 11 compounds that significantly stabilized DDK. Several inhibited DDK with comparable potency to PHA-767491, including Chk1 and PKR kinase inhibitors, but had divergent chemical scaffolds from known DDK inhibitors. Taken together, these data show that several well-known kinase inhibitors cross-react with DDK and also highlight the opportunity to design additional specific, biologically active DDK inhibitors for use as chemotherapeutic agents.
The mammalian telomeric shelterin complex—comprised of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1—blocks the DNA damage response at chromosome ends and interacts with telomerase and the CST complex to regulate telomere length. The evolutionary origins of shelterin are unclear, partly because unicellular organisms have distinct telomeric proteins. Here, we describe the evolution of metazoan shelterin, showing that TRF1 emerged in vertebrates upon duplication of a TRF2-like ancestor. TRF1 and TRF2 diverged rapidly during vertebrate evolution through the acquisition of new domains and interacting factors. Vertebrate shelterin is also distinguished by the presence of an HJRL domain in the split C-terminal OB fold of POT1, whereas invertebrate POT1s carry inserts of variable nature. Importantly, the data reveal that, apart from the primate and rodent POT1 orthologs, all metazoan POT1s are predicted to have a fourth OB fold at their N termini. Therefore, we propose that POT1 arose from a four-OB-fold ancestor, most likely an RPA70-like protein. This analysis provides insights into the biology of shelterin and its evolution from ancestral telomeric DNA-binding proteins.
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