title: ATM and ATR promote Mre11 dependent restart of collapsed replication forks and prevent accumulation of DNA breaks.
abstract: Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia Rad3-related (ATR) and the Mre11/Rad50/Nbs1 complex ensure genome stability in response to DNA damage. However, their essential role in DNA metabolism remains unknown. Here we show that ATM and ATR prevent accumulation of DNA double-strand breaks (DSBs) during chromosomal replication. Replicating chromosomes accumulate DSBs in Xenopus laevis egg extracts depleted of ATM and ATR. Addition of ATM and ATR proteins to depleted extracts prevents DSB accumulation by promoting restart of collapsed replication forks that arise during DNA replication. We show that collapsed forks maintain MCM complex but lose Pol epsilon, and that Pol epsilon reloading requires ATM and ATR. Replication fork restart is abolished in Mre11 depleted extracts and is restored by supplementation with recombinant human Mre11/Rad50/Nbs1 complex. Using a novel fluorescence resonance energy transfer-based technique, we demonstrate that ATM and ATR induce Mre11/Rad50/Nbs1 complex redistribution to restarting forks. This study provides direct biochemical evidence that ATM and ATR prevent accumulation of chromosomal abnormalities by promoting Mre11/Rad50/Nbs1 dependent recovery of collapsed replication forks.
authors.affiliation: Genome Stability Unit, London Research Institute, Clare Hall Laboratories, South Mimms, London, UK.
authors.forename: Kristina
authors.initials: K
authors.lastname: Trenz
authors.forename: Eloise
authors.initials: E
authors.lastname: Smith
authors.forename: Sarah
authors.initials: S
authors.lastname: Smith
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Costanzo
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002474:Cell-Free System; D033481:Chromosomes, Mammalian / Q000502:physiology; D004249:DNA Damage; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology*; D005260:Female; D066298:In Vitro Techniques; D000076228:MRE11 Homologue Protein; D009865:Oocytes / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D029867:Xenopus Proteins / Q000378:metabolism* / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C510917:Mre11 protein, Xenopus; D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins; C576898:Atr protein, Xenopus; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D000076228:MRE11 Homologue Protein