title: The Replication Checkpoint Prevents Two Types of Fork Collapse without Regulating Replisome Stability.
abstract: The ATR replication checkpoint ensures that stalled forks remain stable when replisome movement is impeded. Using an improved iPOND protocol combined with SILAC mass spectrometry, we characterized human replisome dynamics in response to fork stalling. Our data provide a quantitative picture of the replisome and replication stress response proteomes in 32 experimental conditions. Importantly, rather than stabilize the replisome, the checkpoint prevents two distinct types of fork collapse. Unsupervised hierarchical clustering of protein abundance on nascent DNA is sufficient to identify protein complexes and place newly identified replisome-associated proteins into functional pathways. As an example, we demonstrate that ZNF644 complexes with the G9a/GLP methyltransferase at replication forks and is needed to prevent replication-associated DNA damage. Our data reveal how the replication checkpoint preserves genome integrity, provide insights into the mechanism of action of ATR inhibitors, and will be a useful resource for replication, DNA repair, and chromatin investigators.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
authors.forename: Huzefa
authors.initials: H
authors.lastname: Dungrawala
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA; Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
authors.forename: Kristie L
authors.initials: KL
authors.lastname: Rose
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
authors.forename: Kamakoti P
authors.initials: KP
authors.lastname: Bhat
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
authors.forename: Kareem N
authors.initials: KN
authors.lastname: Mohni
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
authors.forename: Gloria G
authors.initials: GG
authors.lastname: Glick
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
authors.forename: Frank B
authors.initials: FB
authors.lastname: Couch
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Electronic address: david.cortez@vanderbilt.edu.
authors.forename: David
authors.initials: D
authors.lastname: Cortez
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D004249:DNA Damage; D004265:DNA Helicases / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D004795:Enzyme Stability; D057809:HEK293 Cells; D006801:Humans; D059807:S Phase Cell Cycle Checkpoints; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D014157:Transcription Factors; C573234:ZNF644 protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D004259:DNA-Directed DNA Polymerase; D003851:Deoxyribonucleases; D004265:DNA Helicases; D045643:DNA Repair Enzymes