title: The Mre11-Rad50-Nbs1 (MRN) complex has a specific role in the activation of Chk1 in response to stalled replication forks.
abstract: The activation of Chk1 in response to stalled replication forks in Xenopus egg extracts involves a complex pathway containing ATM and Rad3-related (ATR), topoisomerase IIβ-binding protein 1 (TopBP1), Rad17, the Rad9-Hus1-Rad1 (9-1-1) complex, and Claspin. We have observed that egg extracts lacking the Mre11-Rad50-Nbs1 (MRN) complex show greatly, although not completely, reduced activation of Chk1 in response to replication blockages. Depletion of both Rad17 and MRN leads to a further, essentially complete, reduction in the activation of Chk1. Thus, Rad17 and MRN act in at least a partially additive manner in promoting activation of Chk1. There was not an obvious change in the binding of RPA, ATR, Rad17, or the 9-1-1 complex to chromatin in aphidicolin (APH)-treated, MRN-depleted extracts. However, there was a substantial reduction in the binding of TopBP1. In structure-function studies of the MRN complex, we found that the Mre11 subunit is necessary for the APH-induced activation of Chk1. Moreover, a nuclease-deficient mutant of Mre11 cannot substitute for wild-type Mre11 in this process. These results indicate that the MRN complex, in particular the nuclease activity of Mre11, plays an important role in the activation of Chk1 in response to stalled replication forks. These studies reveal a previously unknown property of the MRN complex in genomic stability.
authors.affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Joon
authors.initials: J
authors.lastname: Lee
authors.forename: William G
authors.initials: WG
authors.lastname: Dunphy
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002474:Cell-Free System; D000071877:Checkpoint Kinase 1; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007399:Interphase; D000076228:MRE11 Homologue Protein; D046912:Multiprotein Complexes / Q000378:metabolism; D009865:Oocytes; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D061987:Sf9 Cells; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D029867:Xenopus Proteins / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D002352:Carrier Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C510917:Mre11 protein, Xenopus; D046912:Multiprotein Complexes; C511600:NBN protein, Xenopus; C542167:Rad50 protein, Xenopus; D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D000076228:MRE11 Homologue Protein; D000251:Adenosine Triphosphatases; C551927:Rad17 protein, Xenopus; D045643:DNA Repair Enzymes