title: ATM protein physically and functionally interacts with proliferating cell nuclear antigen to regulate DNA synthesis.
abstract: Ataxia telangiectasia (A-T) is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation that is caused by biallelic mutations in A-T mutated (ATM), a gene encoding a protein kinase critical for the induction of cellular responses to DNA damage, particularly to DNA double strand breaks. A long known characteristic of A-T cells is their ability to synthesize DNA even in the presence of ionizing radiation-induced DNA damage, a phenomenon termed radioresistant DNA synthesis. We previously reported that ATM kinase inhibition, but not ATM protein disruption, blocks sister chromatid exchange following DNA damage. We now show that ATM kinase inhibition, but not ATM protein disruption, also inhibits DNA synthesis. Investigating a potential physical interaction of ATM with the DNA replication machinery, we found that ATM co-precipitates with proliferating cell nuclear antigen (PCNA) from cellular extracts. Using bacterially purified ATM truncation mutants and in vitro translated PCNA, we showed that the interaction is direct and mediated by the C terminus of ATM. Indeed, a 20-amino acid region close to the kinase domain is sufficient for strong binding to PCNA. This binding is specific to ATM, because the homologous regions of other PIKK members, including the closely related kinase A-T and Rad3-related (ATR), did not bind PCNA. ATM was found to bind two regions in PCNA. To examine the functional significance of the interaction between ATM and PCNA, we tested the ability of ATM to stimulate DNA synthesis by DNA polymerase δ, which is implicated in both DNA replication and DNA repair processes. ATM was observed to stimulate DNA polymerase activity in a PCNA-dependent manner.
authors.affiliation: Department of Radiation Oncology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213-1863, USA.
authors.forename: Armin M
authors.initials: AM
authors.lastname: Gamper
authors.forename: Serah
authors.initials: S
authors.lastname: Choi
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Matsumoto
authors.forename: Dibyendu
authors.initials: D
authors.lastname: Banerjee
authors.forename: Alan E
authors.initials: AE
authors.lastname: Tomkinson
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Bakkenist
mesh_terms: D020413:3' Untranslated Regions; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D064007:Ataxia Telangiectasia Mutated Proteins; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis*; D004258:DNA Polymerase III / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D013892:Thioxanthenes / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C546193:2-(2,6-dimethylmorpholin-4-yl)-N-(5-(6-morpholin-4-yl-4-oxo-4H-pyran-2-yl)-9H-thioxanthen-2-yl)acetamide; D020413:3' Untranslated Regions; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009025:Morpholines; D010446:Peptide Fragments; D018809:Proliferating Cell Nuclear Antigen; D013892:Thioxanthenes; D025521:Tumor Suppressor Proteins; D004247:DNA; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D004258:DNA Polymerase III