Abstract
The MRN (MRE11-RAD50-NBS1) complex has been implicated in many aspects of the DNA damage response. It has key roles in sensing and processing DNA double-strand breaks, as well as in activation of ATM (ataxia telangiectasia mutated). We reveal a function for MRN in ATR (ATM- and RAD3-related) activation by using defined ATR-activating DNA structures in Xenopus egg extracts. Strikingly, we demonstrate that MRN is required for recruitment of TOPBP1 to an ATR-activating structure that contains a single-stranded DNA (ssDNA) and a double-stranded DNA (dsDNA) junction and that this recruitment is necessary for phosphorylation of CHK1. We also show that the 911 (RAD9-RAD1-HUS1) complex is not required for TOPBP1 recruitment but is essential for TOPBP1 function. Thus, whereas MRN is required for TOPBP1 recruitment at an ssDNA-to-dsDNA junction, 911 is required for TOPBP1 "activation." These findings provide molecular insights into how ATR is activated.
Copyright © 2013 Elsevier Inc. All rights reserved.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Ataxia Telangiectasia Mutated Proteins
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Binding Sites
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Cell Line, Tumor
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Checkpoint Kinase 1
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Chromosomal Proteins, Non-Histone / metabolism
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DNA Repair Enzymes
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DNA, Single-Stranded / chemistry
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DNA, Single-Stranded / metabolism
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism*
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Enzyme Activation
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Humans
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MRE11 Homologue Protein
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Multiprotein Complexes
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Nucleic Acid Conformation
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Phosphorylation
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Protein Binding
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Protein Kinases / metabolism
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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RNA Interference
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Transfection
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Tumor Suppressor Proteins / genetics
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Tumor Suppressor Proteins / metabolism*
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Xenopus Proteins / genetics
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Xenopus Proteins / metabolism*
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Xenopus laevis / genetics
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Xenopus laevis / metabolism*
Substances
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Carrier Proteins
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Cell Cycle Proteins
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Chromosomal Proteins, Non-Histone
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DNA, Single-Stranded
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DNA-Binding Proteins
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Hus1 protein, Xenopus
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Mre11 protein, Xenopus
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Multiprotein Complexes
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NBN protein, Xenopus
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Rad1 protein, Xenopus
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Rad50 protein, Xenopus
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TopBP1 protein, Xenopus
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Tumor Suppressor Proteins
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Xenopus Proteins
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rad9 protein
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Protein Kinases
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Atr protein, Xenopus
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ATM protein, human
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Ataxia Telangiectasia Mutated Proteins
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CHEK1 protein, human
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Checkpoint Kinase 1
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Chek1 protein, Xenopus
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Protein Serine-Threonine Kinases
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MRE11 Homologue Protein
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DNA Repair Enzymes