A DNA Damage-Regulated BRCT-Containing Protein, TopBP1, Is Required for Cell Survival

Abstract

BRCA1 carboxyl-terminal (BRCT) motifs are present in a number of proteins involved in DNA repair and/or DNA damage-signaling pathways. Human DNA topoisomerase II binding protein 1 (TopBP1) contains eight BRCT motifs and shares sequence similarity with the fission yeast Rad4/Cut5 protein and the budding yeast DPB11 protein, both of which are required for DNA damage and/or replication checkpoint controls. We report here that TopBP1 is phosphorylated in response to DNA double-strand breaks and replication blocks. TopBP1 forms nuclear foci and localizes to the sites of DNA damage or the arrested replication forks. In response to DNA strand breaks, TopBP1 phosphorylation depends on the ataxia telangiectasia mutated protein (ATM) in vivo. However, ATM-dependent phosphorylation of TopBP1 does not appear to be required for focus formation following DNA damage. Instead, focus formation relies on one of the BRCT motifs, BRCT5, in TopBP1. Antisense Morpholino oligomers against TopBP1 greatly reduced TopBP1 expression in vivo. Similar to that of ataxia telangiectasia-related protein (ATR), Chk1, or Hus1, downregulation of TopBP1 leads to reduced cell survival, probably due to increased apoptosis. Taken together, the data presented here suggest that, like its putative counterparts in yeast species, TopBP1 may be involved in DNA damage and replication checkpoint controls.Cell cycle checkpoints induced by DNA damage are essential for maintaining genetic integrity. Signals of DNA damage lead to cell cycle arrest and allow time for the repair of damaged DNA (for recent reviews, see references 41, 45, and 72). Failure of checkpoint responses results in genetic instability, frequently leading to cancer development.In mammals, ataxia telangiectasia mutated protein (ATM) and ataxia telangiectasia-related protein (ATR), two phosphatidylinositol-3 kinase (PI3K)-related protein kinases, are essential components in DNA damage-signaling pathways. In response to DNA damage and/or replication blocks, ATM and ATR activate the downstream checkpoint kinases Chk1 and Chk2/Cds1 (see references 41, 45, and 72 for details). Together, these four DNA damage-activated kinases phosphorylate and regulate a number of proteins, including Cdc25C (4,7,13,35,39,51), Cdc25A (21,36), NBS1 (24,34,65,70), p53 (3,11,14,28,31,55,58), BRCA1 (15,17,23,25,32,59), and CtIP (33). By regulating the functions of these proteins and other unidentified substrates, these kinases play essential roles in coordinating DNA repair, cell cycle progression, transcriptional regulation, and apoptosis in response to various DNAdamaging events.In order to understand in detail the mammalian DNA damage-signaling pathway, one has to identify the physiological substrates of ATM and ATR. It is interesting that several ATM and/or ATR substrates, including BRCA1 and NBS1, contain BRCA1 carboxyl-terminal (BRCT) motifs. BRCT motifs were originally identified in the breast cancer tumor suppressor protein BRCA1 (30) and have since been identified in a number of proteins involved in DNA repair (...

Nijmegen breakage syndrome gene, NBS1, and molecular links to factors for genome stability

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The E3 ubiquitin ligase EDD is an adverse prognostic factor for serous epithelial ovarian cancer and modulates cisplatin resistance in vitro

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An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

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