Shengfang Jin

Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae.

DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were also sensitive to methylmethane sulfonate (MMS). Ku-dependent repair is distinct from homologous recombination, because deletion of KU80 and HDF1 increased the MMS sensitivity of rad52delta. Interestingly, rad5Odelta, also shown here to be defective in end joining, was epistatic with Ku mutations for MMS repair and end joining. Therefore, Ku and Rad50 participate in an end-joining pathway that is distinct from homologous recombinational repair. Yeast DNA end joining is functionally analogous to DSB repair and V(D)J recombination in mammalian cells.

Inactivation of DNA-Dependent Protein Kinase by Protein Kinase Cδ: Implications for Apoptosis

ABSTRACT Protein kinase Cδ (PKCδ) is proteolytically cleaved and activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. A role for PKCδ in apoptosis is supported by the finding that overexpression of the catalytic fragment of PKCδ (PKCδ CF) in cells is associated with the appearance of certain characteristics of apoptosis. However, the functional relationship between PKCδ cleavage and induction of apoptosis is unknown. The present studies demonstrate that PKCδ associates constitutively with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The results show that PKCδ CF phosphorylates DNA-PKcs in vitro. Interaction of DNA-PKcs with PKCδ CF inhibits the function of DNA-PKcs to form complexes with DNA and to phosphorylate its downstream target, p53. The results also demonstrate that cells deficient in DNA-PK are resistant to apoptosis induced by overexpressing PKCδ CF. These findings support the hypothesis that functional interactions between PKCδ and DNA-PK contribute to DNA damage-induced apoptosis.

Double-strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions.

Heterodimers of the 70 and 80 kDa Ku autoantigens (Ku70 and Ku80) activate the DNA‐dependent protein kinase (DNA‐PK). Mutations in any of the three subunits of this protein kinase (Ku70, Ku80 and DNA‐PKcs) lead to sensitivity to ionizing radiation (IR) and to DNA double‐strand breaks, and V(D)J recombination product formation defects. Here we show that the IR repair, DNA end binding and DNA‐PK defects in Ku70−/− embryonic stem cells can be counteracted by introducing epitope‐tagged wild‐type Ku70 cDNA. Truncations and chimeras of Ku70 were used to identify the regions necessary for DNA end binding and IR repair. Site‐specific mutational analysis revealed a core region of Ku70 responsible for DNA end binding and heterodimerization. The propensity for Ku70 to associate with Ku80 and to bind DNA correlates with the ability to activate DNA‐PK, although two mutants showed that the roles of Ku70 in DNA‐PK activation and IR repair are separate. Mutation of DNA‐PK autophosphorylation sites and other structural motifs in Ku70 showed that these sites are not necessary for IR repair in vivo. These studies reveal Ku70 features required for double‐strand break repair.

Binding of Ku and c-Abl at the kinase homology region of DNA-dependent protein kinase catalytic subunit.

The DNA-dependent protein kinase (DNA-PK) controls the repair of double-stranded DNA breaks in mammalian cells. The protein kinase subunit of DNA-PK (DNA-PKcs) is targeted to DNA breaks by association with the Ku DNA-binding heterodimer. Here we show that a Ku association site is present at the carboxyl terminus of DNA-PKcs (amino acids 3002–3850) near the protein kinase domain. Correspondingly, the nuclear c-Abl tyrosine kinase that associates with DNA-PK also binds to the kinase homology domain. The c-Abl SH3 domain binds to amino acids 3414–3850 of DNA-PKcs. c-Abl phosphorylates C-terminal fragments of DNA-PKcs, particularly amino acids 3414–3850. c-Abl phosphorylation of DNA-PKcs disassociates the DNA-PKcs·Ku complex. Thus, Ku and c-Abl provide opposing functions with regard to DNA-PK activity.

Regulation of DNA-dependent Protein Kinase by the Lyn Tyrosine Kinase

The Src-like protein-tyrosine kinase Lyn is activated by ionizing radiation and certain other DNA-damaging agents, whereas the DNA-dependent protein kinase (DNA-PK), consisting of the catalytic subunits (DNA-PKcs) and Ku DNA-binding components, requires DNA double-stranded breaks for activation. Here we demonstrate that Lyn associates constitutively with DNA-PKcs. The SH3 domain of Lyn interacts directly with DNA-PKcs near a leucine zipper homology domain. We also show that Lyn phosphorylates DNA-PKcs but not Ku in vitro. The interaction between Lyn and DNA-PKcs inhibits DNA-PKcs activity and the ability of DNA-PKcsto form a complex with Ku/DNA. These results support the hypothesis that there are functional interactions between Lyn and DNA-PKcs in the response to DNA damage.

The IDH2 R172K mutation associated with angioimmunoblastic T-cell lymphoma produces 2HG in T cells and impacts lymphoid development.

Significance Mutations in isocitrate dehydrogenase (IDH)1 and IDH2 contribute to malignant progression by producing the oncometabolite 2HG. In myeloid disorders, mutations at three positions in these genes are commonly observed, but in angioimmunoblastic T-cell lymphoma (AITL), IDH mutations are restricted to IDH2 arginine (R) 172. The complex microenvironment of AITL, where malignant T cells comprise a minority of the tumor, has made it difficult to evaluate the role of this mutation. Here, we provide clinical data showing that mutant IDH2 expression is restricted to malignant T cells and that 2HG may be a useful biomarker in AITL. In addition, using conditional knock-in mouse models, we find that only mutations at IDH2 R172 produce significant quantities of 2HG in lymphoid cells and alter lymphoid development. Oncogenic isocitrate dehydrogenase (IDH)1 and IDH2 mutations at three hotspot arginine residues cause an enzymatic gain of function that leads to the production and accumulation of the metabolite 2-hydroxyglutarate (2HG), which contributes to the development of a number of malignancies. In the hematopoietic system, mutations in IDH1 at arginine (R) 132 and in IDH2 at R140 and R172 are commonly observed in acute myeloid leukemia, and elevated 2HG is observed in cells and serum. However, in angioimmunoblastic T-cell lymphoma (AITL), mutations are almost exclusively restricted to IDH2 R172, and levels of 2HG have not been comprehensively measured. In this study, we investigate the expression pattern of mutant IDH2 in the AITL tumor microenvironment and measure levels of 2HG in tissue and serum of AITL patients. We find that mutant IDH2 expression is restricted to the malignant T-cell component of AITL, and that 2HG is elevated in tumor tissue and serum of patients. We also investigate the differences between the three hotspot mutation sites in IDH1 and IDH2 using conditional knock-in mouse models. These studies show that in the lymphoid system, mutations in IDH2 at R172 produce high levels of 2HG compared with mutations at the other two sites and that lymphoid development is impaired in these animals. These data provide evidence that IDH2 R172 mutations may be the only variants present in AITL because of their capacity to produce significant amounts of the oncometabolite 2HG in the cell of origin of this disease.