Padmini Kedar

Cellular localisation of the ataxia-telangiectasia (ATM) gene product and discrimination between mutated and normal forms.

The recently cloned gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is involved in DNA damage response at different cell cycle checkpoints and also appears to have a wider role in signal transduction. Antibodies prepared against peptides from the predicted protein sequence detected a ∼ 350 kDa protein corresponding to the open reading frame, which was absent in 13/23 A-T homozygotes. Subcellular fractionation, immunoelectronmicroscopy and immunofluorescence showed that the ATM protein is present in the nucleus and cytoplasmic vesicles. This distribution did not change after irradiation. We also provide evidence that ATM protein binds to p53 and this association is defective in A-T cells compatible with the defective p53 response in these cells. These results provide further support for a role for the ATM protein as a sensor of DNA damage and in a more general role in cell signalling, compatible with the broader phenotype of the syndrome.

Localization of a Portion of Extranuclear ATM to Peroxisomes

The gene mutated in the human genetic disorder ataxia-telangiectasia codes for a protein, ATM, the known functions of which include response to DNA damage, cell cycle control, and meiotic recombination. Consistent with these functions, ATM is predominantly present in the nucleus of proliferating cells; however, a significant proportion of the protein has also been detected outside the nucleus in cytoplasmic vesicles. To understand the possible role of extra-nuclear ATM, we initially investigated the nature of these vesicles. In this report we demonstrate that a portion of ATM co-localizes with catalase, that ATM is present in purified mouse peroxisomes, and that there are reduced levels of ATM in the post-mitochondrial membrane fraction of cells from a patient with a peroxisome biogenesis disorder. Furthermore the use of the yeast two-hybrid system demonstrated that ATM interacts directly with a protein involved in the import of proteins into the peroxisome matrix. Because peroxisomes are major sites of oxidative metabolism, we investigated catalase activity and lipid hydroperoxide levels in normal and A-T fibroblasts. Significantly decreased catalase activity and increased lipid peroxidation was observed in several A-T cell lines. The localization of ATM to peroxisomes may contribute to the pleiotropic nature of A-T.

Coordination of Steps in Single-nucleotide Base Excision Repair Mediated by Apurinic/Apyrimidinic Endonuclease 1 and DNA Polymerase β

The individual steps in single-nucleotide base excision repair (SN-BER) are coordinated to enable efficient repair without accumulation of cytotoxic DNA intermediates. The DNA transactions and various proteins involved in SN-BER of abasic sites are well known in mammalian systems. Yet, despite a wealth of information on SN-BER, the mechanism of step-by-step coordination is poorly understood. In this study we conducted experiments toward understanding step-by-step coordination during BER by comparing DNA binding specificities of two major human SN-BER enzymes, apurinic/aprymidinic endonuclease 1 (APE) and DNA polymerase β (Pol β). It is known that these enzymes do not form a stable complex in solution. For each enzyme, we found that DNA binding specificity appeared sufficient to explain the sequential processing of BER intermediates. In addition, however, we identified at higher enzyme concentrations a ternary complex of APE·Pol β·DNA that formed specifically at BER intermediates containing a 5′-deoxyribose phosphate group. Formation of this ternary complex was associated with slightly stronger Pol β gap-filling and much stronger 5′-deoxyribose phosphate lyase activities than was observed with the Pol β·DNA binary complex. These results indicate that step-by-step coordination in SN-BER can rely on DNA binding specificity inherent in APE and Pol β, although coordination also may be facilitated by APE·Pol β·DNA ternary complex formation with appropriate enzyme expression levels or enzyme recruitment to sites of repair.

Increased PARP-1 association with DNA in alkylation damaged, PARP-inhibited mouse fibroblasts

Treatment of base excision repair–proficient mouse fibroblasts with the DNA alkylating agent methyl methanesulfonate (MMS) and a small molecule inhibitor of PARP-1 results in a striking cell killing phenotype, as previously reported. Earlier studies showed that the mechanism of cell death is apoptosis and requires DNA replication, expression of PARP-1, and an intact S-phase checkpoint cell signaling system. It is proposed that activity-inhibited PARP-1 becomes immobilized at DNA repair intermediates, and that this blocks DNA repair and interferes with DNA replication, eventually promoting an S-phase checkpoint and G2-M block. Here we report studies designed to evaluate the prediction that inhibited PARP-1 remains DNA associated in cells undergoing repair of alkylation-induced damage. Using chromatin immunoprecipitation with anti–PARP-1 antibody and qPCR for DNA quantification, a higher level of DNA was found associated with PARP-1 in cells treated with MMS plus PARP inhibitor than in cells without inhibitor treatment. These results have implications for explaining the extreme hypersensitivity phenotype after combination treatment with MMS and a PARP inhibitor. Mol Cancer Res; 10(3); 360–8. ©2012 AACR.

Approaches to determine the specific role of the delta isoform of protein kinase C

Two dimensional gel electrophoresis of proteins from HL-60 human leukaemia cells treated with bistratene A, a specific activator of protein kinase C (PKC) delta, was performed in conjunction with sequencing in order to identify components of the signal transduction pathway of this isoform of PKC. Stathmin (oncoprotein 18) was identified in this way and the phosphorylation of this protein after treatment with bistratene A, was confirmed by Western blotting of 2D gels. Since stathmin has phosphorylation sites for mitogen activated protein (MAP) kinases, cyclin dependent kinases and calcium/calmodulin dependent protein kinases, it is assumed that one of these enzymes, acting downstream from PKC delta, is responsible for the phosphorylation. Another approach to determining the role of PKC delta involves the identification of interacting proteins using the yeast two hybrid screen. The sequence of nine out of ten independently isolated clones from a two hybrid screen showed perfect homology to human ribosomal protein L8. This protein has previously been shown to exist in complexes with ribosomal RNA, aminoacyl-tRNA and elongation factor-1 alpha, a known substrate of PKC delta, suggesting a role for PKC delta in protein synthesis regulation.