title: S-phase-dependent p50/NF-кB1 phosphorylation in response to ATR and replication stress acts to maintain genomic stability.
abstract: The apical damage kinase, ATR, is activated by replication stress (RS) both in response to DNA damage and during normal S-phase. Loss of function studies indicates that ATR acts to stabilize replication forks, block cell cycle progression and promote replication restart. Although checkpoint failure and replication fork collapse can result in cell death, no direct cytotoxic pathway downstream of ATR has previously been described. Here, we show that ATR directly reduces survival by inducing phosphorylation of the p50 (NF-κB1, p105) subunit of NF-кB and moreover, that this response is necessary for genome maintenance independent of checkpoint activity. Cell free and in vivo studies demonstrate that RS induces phosphorylation of p50 in an ATR-dependent but DNA damage-independent manner that acts to modulate NF-кB activity without affecting p50/p65 nuclear translocation. This response, evident in human and murine cells, occurs not only in response to exogenous RS but also during the unperturbed S-phase. Functionally, the p50 response results in inhibition of anti-apoptotic gene expression that acts to sensitize cells to DNA strand breaks independent of damage repair. Ultimately, loss of this pathway causes genomic instability due to the accumulation of chromosomal breaks. Together, the data indicate that during S-phase ATR acts via p50 to ensure that cells with elevated levels of replication-associated DNA damage are eliminated.
authors.affiliation: a Department of Surgery, Section of Neurosurgery ; The University of Chicago ; Chicago , IL USA.
authors.forename: Clayton D
authors.initials: CD
authors.lastname: Crawley
authors.forename: Shijun
authors.initials: S
authors.lastname: Kang
authors.forename: Giovanna M
authors.initials: GM
authors.lastname: Bernal
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Wahlstrom
authors.forename: David J
authors.initials: DJ
authors.lastname: Voce
authors.forename: Kirk E
authors.initials: KE
authors.lastname: Cahill
authors.forename: Andrea
authors.initials: A
authors.lastname: Garofalo
authors.forename: David R
authors.initials: DR
authors.lastname: Raleigh
authors.forename: Ralph R
authors.initials: RR
authors.lastname: Weichselbaum
authors.forename: Bakhtiar
authors.initials: B
authors.lastname: Yamini
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D020552:Comet Assay; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D042822:Genomic Instability / Q000235:genetics / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008156:Luciferases; D052002:NF-kappa B p50 Subunit / Q000378:metabolism*; D010766:Phosphorylation; D034622:RNA Interference; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase / Q000502:physiology*
chemical_list: D017931:DNA Primers; D052002:NF-kappa B p50 Subunit; D008156:Luciferases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins
keywords: ATM, ataxia telangiectasia mutated; ATR; ATR, ataxia telangiectasia mutated and Rad3-related; Bax, BCL2-associated X protein; Bclxl, Bcl-2-like protein; ChIP, chromatin immunoprecipitation; Chk1, checkpoint kinase 1; DNA damage; DSBs, double-strand breaks; H2AX, histone 2AX; HR, homologous recombination; Hu, hydroxyurea; IR, ionizing radiation; IκB, inhibitor kappaB; IκK, inhibitor kappaB kinase; NF-κB; NF-κB, nuclear factor-kappaB; RS, replication stress; RT-PCR, reverse transcriptase polymerase chain reaction; S-phase; TAM, tamoxifen; TMZ, temozolomide; TopBP1, topoisomerase-binding protein-1; p50; replication stress; siRNA, short interfering RNA