title: Cdc45 is limiting for replication initiation in humans.
abstract: Cdc45 is an essential protein that together with Mcm2-7 and GINS forms the eukaryotic replicative helicase CMG. Cdc45 seems to be rate limiting for the initial unwinding or firing of replication origins. In line with this view, Cdc45-overexpressing cells fired at least twice as many origins as control cells. However, these cells displayed an about 2-fold diminished fork elongation rate, a pronounced asymmetry of replication fork extension, and an early S phase arrest. This was accompanied by H2AX-phosphorylation and subsequent apoptosis. Unexpectedly, we did not observe increased ATR/Chk1 signaling but rather a mild ATM/Chk2 response. In addition, we detected accumulation of long stretches of single-stranded DNA, a hallmark of replication catastrophe. We conclude that increased origin firing by upregulated Cdc45 caused exhaustion of the single-strand binding protein RPA, which in consequence diminished the ATR/Chk1 response; the subsequently occurring fork breaks led to an ATM/Chk2 mediated phosphorylation of H2AX and eventually to apoptosis.
authors.affiliation: a Research group Biochemistry, Leibniz Institute for Age Research - Fritz Lipmann Institute , Jena , Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Köhler
authors.affiliation: a Research group Biochemistry, Leibniz Institute for Age Research - Fritz Lipmann Institute , Jena , Germany.
authors.forename: Dennis
authors.initials: D
authors.lastname: Koalick
authors.affiliation: a Research group Biochemistry, Leibniz Institute for Age Research - Fritz Lipmann Institute , Jena , Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Fabricius
authors.affiliation: b Laboratory of Radiobiology and Experimental Radiation Oncology, University Medical Center Hamburg-Eppendorf , Hamburg , Germany.
authors.forename: Ann Christin
authors.initials: AC
authors.lastname: Parplys
authors.affiliation: b Laboratory of Radiobiology and Experimental Radiation Oncology, University Medical Center Hamburg-Eppendorf , Hamburg , Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Borgmann
authors.affiliation: a Research group Biochemistry, Leibniz Institute for Age Research - Fritz Lipmann Institute , Jena , Germany.|c Faculty of Biochemistry and Molecular Medicine, University of Oulu , Finland.
authors.forename: Helmut
authors.initials: H
authors.lastname: Pospiech
authors.affiliation: a Research group Biochemistry, Leibniz Institute for Age Research - Fritz Lipmann Institute , Jena , Germany.|d Centre for Molecular Biomedicine, Friedrich-Schiller University , Jena , Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Grosse
mesh_terms: D017209:Apoptosis; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000032:analysis; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D018741:Replication Origin; D059807:S Phase Cell Cycle Checkpoints; D015398:Signal Transduction
chemical_list: C113571:CDC45 protein, human; D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; C468783:H2AX protein, human; D006657:Histones
keywords: Apoptosis; CMG helicase; RPA; genome instability; origin firing; replication catastrophe; single-strand DNA