The DNA-dependent protein kinase (DNA-PK) is a heterotrimeric enzyme that binds to double-stranded DNA and is required for the rejoining of double-stranded DNA breaks in mammalian cells. It has been proposed that DNA-PK functions in this DNA repair pathway by binding to the ends of broken DNA molecules and phosphorylating proteins that bind to the damaged DNA ends. Another enzyme that binds to DNA strand breaks and may also function in the cellular response to DNA damage is the poly(ADP-ribose) polymerase (PARP). Here, we show that PARP can be phosphorylated by purified DNA-PK, and the catalytic subunit of DNA-PK is ADP-ribosylated by PARP. The protein kinase activity of DNA-PK can be stimulated by PARP in the presence of NAD ؉ in a reaction that is blocked by the PARP inhibitor 1,5-dihydroxyisoquinoline. The stimulation of DNA-PK by PARP-mediated protein ADP-ribosylation occurs independent of the Ku70/80 complex. Taken together, these results show that PARP can modify the activity of DNA-PK in vitro and suggest that these enzymes may function coordinately in vivo in response to DNA damage.Biochemical pathways that function in the recognition and repair of DNA damage are critical for maintaining genomic integrity. Potentially devastating DNA damage in the form of double-strand breaks (DSBs) 1 can occur when cells are exposed to ionizing radiation, oxidative stress and radiomimetic drugs. The DNA-dependent protein kinase (DNA-PK) is a key component of DNA DSB rejoining pathways in mammalian cells. DNA-PK is a heterotrimeric enzyme complex comprised of a 460-kDa catalytic subunit (1) and a regulatory component consisting of the Ku70 and Ku80 proteins (2, 3). The Ku70 and Ku80 proteins form a heterodimeric complex that binds to the ends of double-stranded DNA with high affinity (4 -8). The catalytic subunit of DNA-PK (DNA-PKcs) binds to the Ku70/80 complex in the presence of double-stranded DNA (9) and phosphorylates a wide variety of protein substrates in vitro on serine and threonine residues (10, 11). The protein kinase activity of DNA-PK is autoregulatory; in the absence of a phosphorylation substrate, DNA-PKcs autophosphorylates and dissociates from the Ku70/80-DNA complex (12).Evidence that DNA-PK plays an integral role in the repair of DNA DSB has been provided through the characterization of rodent cell lines that have mutations that disrupt the expression of the Ku80 (13-19) or DNA-PKcs (20 -24). Although it is clear that DNA-PK is an important component of mammalian DNA DSB repair pathways, it is not known how the enzyme participates in these processes. In vitro, DNA-PK preferentially phosphorylates protein substrates that co-localize on the same DNA molecule (3,25). This suggests that the specificity of the phosphorylation reaction may be regulated, in part, via the co-localization of the enzyme and substrate target on DNA. Based on these data, it has been proposed that DNA-PK could participate in the DNA rejoining reaction by phosphorylating DNA repair factors that co-localize with it on broken DNA en...

Abstract. The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene. (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain-deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to fivefold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most ot-mannosidase to mature forms, however, "~ 20-25 % of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular c~-rnannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of ot-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wildtype cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.

Biological ice nuclei (active at approximately-40C) were extracted from cells of the lichen Rhizoplaca chrysokuca by sonication. Sensitivity to proteases, guanidine hydrochloride, and urea showed these nuclei to be proteinaceous. The nuclei were relatively heat stable, active from pH 1.5 to 12, and active without lipids, thereby demonstrating significant differences from bacterial ice nuclei.It has recently been discovered that many species of lichens (symbiotic associations of fungi and algae) are capable of nucleating ice at relatively warm temperatures, (7). This activity is similar to the ability of certain strains of several species of epiphytic bacteria (e.g., Pseudomonas syringae, Pseudomonas fluorescens, and Erwinia herbicola) to initiate freezing of supercooled water at temperatures as warm as -2C (11, 12). Field-collected lichens which have been found to have ice nucleation activity (INA) at temperatures above -5°C include the genera Rhizoptaca, Xanthoparmelia, and,Xanthoria. Of these, Rhizoplaca chrysoleuca has the warrhest INA temperature (-2.3°C). Attempts to isolate bacteria with INA from lichens have been unsuccessful, suggesting a nonbacterial source of INA. An axenic culture of the lichen fungus R. chrysoleuca showed warmtemperature INA (-2°C), further indicating a nonbacterial source of lichen INA and implicating the fungal partner as the producer of lichen ice nuclei (7). In a survey of axenic cultures of lichen fungi (mycobionts) and lichen algae (photobionts), many of the mycobionts exhibited INA at temperatures above -5°C, but none of the photobionts was an active nucleator above -5°C, providing further evidence that INA in the symbiotic association is due s'olely to the fungal partner (8).The process of isolating and characterizing biological ice nuclei is made difficult by the low numbers of nucleator molecules per cell (15,16,19); however, this difficulty is partially offset by the ease of detecting and quantifying INA. Bacterial ice nuclei from P. syringae have been shown to be proteins located in the gram-negative outer membrane (13).These proteins are thought to consist of monomers which have molecular weights of approximately 150,000 and which aggregate to form larger nucleation sites, and. the INA temperature appears to be directly related to the overall size of these aggregated protein monomers (4, 22). Similar protein monomers which mediate INA in P. fluorescens have been found to have molecular weights of.180,000 (1). Membrane-bound bacterial nuclei are active. only when associated with membrane lipids, as shown.by the fact that delipidating agents such as phospholipases or nonpolar solvents eliminate warm-temperature INA in .these organisms (3). The ice-nucleating bacterium E. herbicola releases membrane vesicles with associated INA (17). Inactivation of bacterial INA by lectins has been reported, suggesting that carbohydrate groups are also involved in ice nucleation sites (9, 10). Bacterial INA is known to be sensitive to heat (14) is inactivated by pHs below 5 (10). It has a...