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. Ku80 associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80⌬, hdf1⌬, or ku80⌬ hdf1⌬ 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 ku80⌬, hdf1⌬, or ku80⌬ hdf1⌬ 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 ku80⌬ and hdf1⌬ 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 rad52⌬. Interestingly, rad50⌬, 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.DNA repair is essential for the successful maintenance and propagation of genetic information. Chromosomal doublestrand breaks (DSBs) may occur spontaneously, during DNA recombination events, or may be induced by DNA damage. In eukaryotes, two major DSB repair pathways have been identified that differ in the requirements for DNA homology. DSB repair by homologous recombination results in the precise repair of the DNA lesion but requires the presence of homologous sequences elsewhere in the genome (e.g., a homologous chromosome or a sister chromatid). This is the primary mechanism of DSB repair in yeast species and prokaryotes. In contrast, higher eukaryotes appear to favor a nonhomologous DNA end-joining pathway for DSB repair. In this case, the two ends of a DSB are joined by a process that is largely independent of terminal DNA sequence homology and that therefore produces junctions that can vary in their sequence composition. In mammalian cells, DSBs are generated as intermediates in immunoglobulin or T-cell receptor gene rearrangement [V(D)J recombination] and are the potentially lethal DNA lesions caused by ionizing radiation (IR) (44). The DNA sequence of these repaired DSBs indicates that the latter pathway is utilized.The three subunits of the DNA-dependent protein kinase (DNA-PK) are needed for both IR repair and V(D)J recombination, as demonstrated by a series of mutational and biochemical experiments. The DNA binding subunit of DNA-PK, named Ku, is a heterodimer of 70-and 80-kDa subunits (Ku70 and Ku80, respectively). Ku avidly bi...

Diphthamide, a posttranslational modification of translation elongation factor 2 that is conserved in all eukaryotes and archaebacteria and is the target of diphtheria toxin, is formed in yeast by the actions of five proteins, Dph1 to -5, and a still unidentified amidating enzyme. Dph2 and Dph5 were previously identified. Here, we report the identification of the remaining three yeast proteins (Dph1, -3, and -4) and show that all five Dph proteins have either functional (Dph1, -2, -3, and -5) or sequence (Dph4) homologs in mammals. We propose a unified nomenclature for these proteins (e.g., HsDph1 to -5 for the human proteins) and their genes based on the yeast nomenclature. We show that Dph1 and Dph2 are homologous in sequence but functionally independent. The human tumor suppressor gene OVCA1, previously identified as homologous to yeast DPH2, is shown to actually be HsDPH1. We show that HsDPH3 is the previously described human diphtheria toxin and Pseudomonas exotoxin A sensitivity required gene 1 and that DPH4 encodes a CSL zinc finger-containing DnaJ-like protein. Other features of these genes are also discussed. The physiological function of diphthamide and the basis of its ubiquity remain a mystery, but evidence is presented that Dph1 to -3 function in vivo as a protein complex in multiple cellular processes.

Saccharomyes cerevisiae rad52 mutants are characterized by severe defects in double-strand break (DSB) repair and recombination. In this study we have identified several regions of RAD52 that are required for these biological functions. We cloned and characterized a RAD52 homolog from Kluyveromyces lactis that partially complemented S. cerevisiae rad52 mutants while exhibiting negative dominance in wild-type (RAD52) strains. The dominant negative effect was suppressed by overexpression of RAD51, an additional gene known to be required for DSB repair and recombination, indicating a genetic interaction between these loci. Furthermore, GAL4 two-hybrid analysis revealed a physical interaction between Rad51 and the carboxy-terminal one-third of Rad52. Deletion alleles of tad52 (with or without the Rad51 association domain) also produced dominant negative defects, suggesting the disruption of repair through nonfunctional interactions with other DSB repair and recombination proteins. RAD51 relieved the negative dominance of each of these alleles either by competitive titration or functional activation of mutant or heterologous Rad52 proteins. These results demonstrate the importance of Rad52-RadS1 interactions and point to the formation of a higher order repair/recombination complex potentially containing other yet unidentified components.

When Furchgott, Murad, and Ignarro were honored with the Nobel prize for the identification of nitric oxide (NO) in 1998, the therapeutic implications of this discovery could not be fully anticipated. This was due to the fact that available therapeutics like NO donors did not allow a constant and long-lasting cyclic guanylyl monophosphate (cGMP) stimulation and had a narrow therapeutic window. Now, 20 years later, the stimulator of soluble guanylate cyclase (sGC), riociguat, is on the market and is the only drug approved for the treatment of two forms of pulmonary hypertension (PAH/CTEPH), and a variety of other sGC stimulators and sGC activators are in preclinical and clinical development for additional indications. The discovery of sGC stimulators and sGC activators is a milestone in the field of NO/sGC/cGMP pharmacology. The sGC stimulators and sGC activators bind directly to reduced, heme-containing and oxidized, heme-free sGC, respectively, which results in an increase in cGMP production. The action of sGC stimulators at the heme-containing enzyme is independent of NO but is enhanced in the presence of NO whereas the sGC activators interact with the heme-free form of sGC. These highly innovative pharmacological principles of sGC stimulation and activation seem to have a very broad therapeutic potential. Therefore, in both academia and industry, intensive research and development efforts have been undertaken to fully exploit the therapeutic benefit of these new compound classes. Here we summarize the discovery of sGC stimulators and sGC activators and the current developments in both compound classes, including the mode of action, the chemical structures, and the genesis of the terminology and nomenclature. In addition, preclinical studies exploring multiple aspects of their in vitro, ex vivo, and in vivo pharmacology are reviewed, providing an overview of multiple potential applications. Finally, the clinical developments, investigating the treatment potential of these compounds in various diseases like heart failure, diabetic kidney disease, fibrotic diseases, and hypertension, are reported. In summary, sGC stimulators and sGC activators have a unique mode of action with a broad treatment potential in cardiovascular diseases and beyond.