In higher eukaryotes, the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) checkpoint kinases play distinct, but partially overlapping, roles in DNA damage response. Yet their interrelated function has not been defined for telomere maintenance. We discover in Drosophila that the two proteins control partially redundant pathways for telomere protection: the loss of ATM leads to the fusion of some telomeres, whereas the loss of both ATM and ATR renders all telomeres susceptible to fusion. The ATM-controlled pathway includes the Mre11 and Nijmegen breakage syndrome complex but not the Chk2 kinase, whereas the ATR-regulated pathway includes its partner ATR-interacting protein but not the Chk1 kinase. This finding suggests that ATM and ATR regulate different molecular events at the telomeres compared with the sites of DNA damage. This compensatory relationship between ATM and ATR is remarkably similar to that observed in yeast despite the fact that the biochemistry of telomere elongation is completely different in the two model systems. We provide evidence suggesting that both the loading of telomere capping proteins and normal telomeric silencing requires ATM and ATR in Drosophila and propose that ATM and ATR protect telomere integrity by safeguarding chromatin architecture that favors the loading of telomere-elongating, capping, and silencing proteins.Mre11 and Nbs1 ͉ telomerase-independent mechanism ͉ telomere capping ͉ telomeric silencing
Association of the highly conserved heterochromatin protein, HP1, with the specialized chromatin of centromeres and telomeres requires binding to a specific histone H3 modification of methylation on lysine 9. This modification is catalyzed by the Drosophila Su(var)3-9 gene product and its homologues. Specific DNA binding activities are also likely to be required for targeting this activity along with HP1 to specific chromosomal regions. The Drosophila HOAP protein is a DNA-binding protein that was identified as a component of a multiprotein complex of HP1 containing Drosophila origin recognition complex (ORC) subunits in the early Drosophila embryo. Here we show direct physical interactions between the HOAP protein and HP1 and specific ORC subunits. Two additional HP1-like proteins (HP1b and HP1c) were recently identified in Drosophila, and the unique chromosomal distribution of each isoform is determined by two independently acting HP1 domains (hinge and chromoshadow domain) (47). We find heterochromatin protein 1/origin recognition complex-associated protein (HOAP) to interact specifically with the originally described predominantly heterochromatic HP1a protein. Both the hinge and chromoshadow domains of HP1a are required for its interaction with HOAP, and a novel peptide repeat located in the carboxyl terminus of the HOAP protein is required for the interaction with the HP1 hinge domain. Peptides that interfere with HP1a/HOAP interactions in co-precipitation experiments also displace HP1 from the heterochromatic chromocenter of polytene chromosomes in larval salivary glands. A mutant for the HOAP protein also suppresses centric heterochromatin-induced silencing, supporting a role for HOAP in centric heterochromatin.
The ubiquitous m-and -calpains are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. Previously, -calpain was found to be enriched in mitochondrial fractions isolated from rat cerebral cortex and SH-SY5Y neuroblastoma cells, but the submitochondrial localization of -calpain was not determined. In the present study, submitochondrial fractionation and digitonin permeabilization studies indicated that both calpain 1 and calpain small subunit 1, which together form -calpain, are present in the mitochondrial intermembrane space. The N terminus of calpain 1 contains an amphipathic ␣-helical domain, and is distinct from the N terminus of calpain 2. Calpain 1, but not calpain 2, was imported into mitochondria. Removal of the N-terminal 22 amino acids of calpain 1 blocked the mitochondrial calpain import, while addition of this N-terminal region to calpain 2 or green fluorescent protein enabled mitochondrial import. The N terminus of calpain 1 was not processed following mitochondrial import, but was removed by autolysis following calpain activation. Calpain small subunit 1 was not directly imported into mitochondria, but was imported in the presence of calpain 1. The presence of a mitochondrial targeting sequence in the N-terminal region of calpain 1 is consistent with the localization of -calpain to the mitochondrial intermembrane space and provides new insight into the possible functions of this cysteine protease.Calpains (EC 3.4.22.17) are a family of Ca 2ϩ -activated cysteine proteases, including both ubiquitous and tissue-specific isoforms, that cleave their substrate proteins at discrete sites to modulate activity (1-3). The best characterized, and the predominant calpains in the central nervous system, are the classical m-and -calpains. Their physiological roles have not been fully elucidated but include cell motility, cell differentiation, membrane fusion, platelet activation, and signal transduction (3). Also extensively investigated have been the pathological roles of calpains in cell death, where calpains can cleave key structural proteins and contribute to the release of death-related proteins such as apoptosis-inducing factor (AIF) 3 (4 -9). At present, it is unclear whether the -and m-calpains have distinct or overlapping functions. They are each heterodimers consisting of a unique 80-kDa large catalytic subunit (calpain 1 or 2) and a common 28-kDa small regulatory subunit (calpain small subunit 1 or 2) (2). In vitro, the substrates of m-and -calpains are similar, if not identical (10). Knock-out of the -calpain large subunit, calpain 1, results in viable mice with reduced platelet aggregation and impaired tyrosine phosphorylation in platelets, but not overt phenotype (11). Knock-out of the m-calpain large subunit, calpain 2, or of calpain small subunit 1 (CSS1) is embryonically lethal (12)(13)(14).Both m-and -calpains are considered to be cytosolic enzymes (2,3, 15,16). An association of m-and -calpains with subcellular organelles including endoplasmic ret...
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