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Hypoxia, a hallmark feature of the tumor microenvironment, causes resistance to conventional chemotherapy, but was recently reported to synergize with poly(ADP-ribose) polymerase inhibitors (PARPis) in homologous recombination-proficient (HR-proficient) cells through suppression of HR. While this synergistic killing occurs under severe hypoxia (, Introduction Cytotoxic therapies have long been the backbone of cancer therapy, as they represent an effective means of killing large numbers of tumor cells. A common problem with cytotoxic therapies [...]

Juvenile ciliopathy syndromes that are associated with renal cysts and premature renal failure are commonly the result of mutations in the gene encoding centrosomal protein CEP290. In addition to centrosomes and the transition zone at the base of the primary cilium, CEP290 also localizes to the nucleus; however, the nuclear function of CEP290 is unknown. Here, we demonstrate that reduction of cellular CEP290 in primary human and mouse kidney cells as well as in zebrafish embryos leads to enhanced DNA damage signaling and accumulation of DNA breaks ex vivo and in vivo. Compared with those from WT mice, primary kidney cells from Cep290-deficient mice exhibited supernumerary centrioles, decreased replication fork velocity, fork asymmetry, and increased levels of cyclin-dependent kinases (CDKs). Treatment of Cep290-deficient cells with CDK inhibitors rescued DNA damage and centriole number. Moreover, the loss of primary cilia that results from CEP290 dysfunction was rescued in 3D cell culture spheroids of primary murine kidney cells after exposure to CDK inhibitors. Together, our results provide a link between CEP290 and DNA replication stress and suggest CDK inhibition as a potential treatment strategy for a wide range of ciliopathy syndromes., Introduction Mutations in CEP290 are known to cause the entire spectrum of ciliopathies, including childhood renal failure due to nephronophthisis (NPHP) (1), Joubert syndrome (JS; MIM 610188), the lethal Meckel [...]

Stalled replication forks activate and are stabilized by the ATR (ataxia-telangiectasia mutated and Rad3 related)-mediated checkpoint, but ultimately, they must also recover from the arrest. Although primed single-stranded DNA (ssDNA) is sufficient for checkpoint activation, it is still unknown how this signal is generated at a stalled replication fork. Furthermore, it is not clear how recovery and fork restart occur in higher eukaryotes. Using Xenopus laevis egg extracts, we show that DNA replication continues at a stalled fork through the synthesis and elongation of new primers independent of the checkpoint. This synthesis is dependent on the activity of proliferating cell nuclear antigen, Pol-[delta], and Pol-[epsilon], and it contributes to the phosphorylation of Chk1. We also used defined DNA structures to show that for a fixed amount of ssDNA, increasing the number of primer--template junctions strongly enhances Chk1 phosphorylation. These results suggest that new primers are synthesized at stalled replication forks by the leading and lagging strand polymerases and that accumulation of these primers may contribute to checkpoint activation. doi/10.1083/jcb.200909105

The genome integrity is maintained by the damage signals produced by ATR-mediated checkpoint activation. The studies on Xenopus egg extracts shows that phosphorylation of Chk1 is triggered by single-stranded DNA (ssDNA) although it cannot carry out the activation independently.

Lesions induced by UV irradiation and cis-platinum cause the functional uncoupling of MCM helicase and DNA polymerase activities. Functional uncoupling of MCM helicase and DNA polymerase activities occurs in response to multiple forms of DNA damage and that there is general mechanism for generation of the checkpoint-activating signal following DNA damage.

A requirement for replication in activation of the ATM- and Rad3-related (ATR)-dependent DNA damage checkpoint is discussed. The involvement of DNA replication in activation of the DNA damage checkpoint was investigated using the Xenopus egg extract system. DNA damage was found to slow replication in a checkpoint-independent way and to be accompanied by replication-dependent recruitment of Rad1 and ATR to chromatin.

ATR [ataxia-telangiectasia-mutated (ATM)- and Rad3-related] is a protein kinase required for both DNA damage-induced cell cycle checkpoint responses and the DNA replication checkpoint that prevents mitosis before the completion of DNA synthesis. Although ATM and ATR kinases share many substrates, the different phenotypes of ATM- and ATR-deficient mice indicate that these kinases are not functionally redundant. Here we demonstrate that ATR but not ATM phosphorylates the human Rad17 (hRad17) checkpoint protein on [Ser.sup.635] and [Ser.sup.645] in vitro. In undamaged synchronized human cells, these two sites were phosphorylated in late [G.sup.1], S, and [G.sup.2]/M, but not in early-mid [G.sup.1]. Treatment of cells with genotoxic stress induced phosphorylation of hRad17 in cells in early-mid [G.sup.1]. Expression of kinase-inactive ATR resulted in reduced phosphorylation of these residues, but these same serine residues were phosphorylated in ionizing radiation (IR)-treated ATM-deficient human cell lines. IR-induced phosphorylation of hRad17 was also observed in ATM-deficient tissues, but induction of [Ser.sup.645] was not optimal. Expression of a hRad17 mutant, with both serine residues changed to alanine, abolished IR-induced activation of the [G.sup.1]/S checkpoint in MCF-7 cells. These results suggest ATR and hRad17 are essential components of a DNA damage response pathway in mammalian cells.

Inappropriate cellular inflammation can cause disease. It emerges that the protein SAMHD1 prevents the release of newly replicated DNA from the nucleus, blocking an undesirable pro-inflammatory response. Inappropriate cellular inflammation can cause disease. It emerges that the protein SAMHD1 prevents the release of newly replicated DNA from the nucleus, blocking an undesirable pro-inflammatory response., Author(s): Madzia P. Crossley, Karlene A. Cimprich Author Affiliations: Faulty replication can sting Cells need to distinguish between their own DNA and that of viruses. To solve this problem, plant, [...]

A family of proteins involved in cell cycle progression, DNA recombination, and the detection of DNA damage has been recently identified. One of the members of this family, human ATM, is defective in the cells of patients with ataxia telangiectasia and is involved in detection and response of cells to damaged DNA. Other members include Mei-41 (Drosophila melanogaster), Mec1p (Saccharomyces cerevisiae), and Rad3 (Schizosaccharomyces pombe), which are required for the S and [G.sub.2]/M checkpoints, as well as FRAP (Homo sapiens) and Tor1/2p (S. cerevisiae), which are involved in a rapamycin-sensitive pathway leading to [G.sub.1] cell cycle progression. We report here the cloning of a human cDNA encoding a protein with significant homology to members of this family. Three overlapping clones isolated from a Jurkat T-cell cDNA library revealed a 7.9-kb open reading frame encoding a protein that we have named FRP1 (FRAP-related protein) with 2644 amino acids and a predicted molecular mass of 301 kDa. Using fluorescence in situ hybridization and a full-length cDNA FRP1 clone, the FRP1 gene has been mapped to the chromosomal locus 3q22-q24. FRP1 is most closely related to three of the PIK-related kinase family members involved in checkpoint function - Mei-41, Meclp, and Rad3 - and as such may be the functional human counterpart of these proteins.

Addition of dialkyl- and alkenylzinc compounds to aldehydes with chiral oxazaborolidines results in better enantioselectivity than nucleophilic alkynylation synthesis of carbonyl compounds. Dimethylborane, an alkynylation reagent, forms propargyl carbinols with good enantioselective properties when added to an aldehyde with chiral oxazaborolidines as catalysts.