title: MRX protects fork integrity at protein-DNA barriers, and its absence causes checkpoint activation dependent on chromatin context.
abstract: To address how eukaryotic replication forks respond to fork stalling caused by strong non-covalent protein-DNA barriers, we engineered the controllable Fob-block system in Saccharomyces cerevisiae. This system allows us to strongly induce and control replication fork barriers (RFB) at their natural location within the rDNA. We discover a pivotal role for the MRX (Mre11, Rad50, Xrs2) complex for fork integrity at RFBs, which differs from its acknowledged function in double-strand break processing. Consequently, in the absence of the MRX complex, single-stranded DNA (ssDNA) accumulates at the rDNA. Based on this, we propose a model where the MRX complex specifically protects stalled forks at protein-DNA barriers, and its absence leads to processing resulting in ssDNA. To our surprise, this ssDNA does not trigger a checkpoint response. Intriguingly, however, placing RFBs ectopically on chromosome VI provokes a strong Rad53 checkpoint activation in the absence of Mre11. We demonstrate that proper checkpoint signalling within the rDNA is restored on deletion of SIR2. This suggests the surprising and novel concept that chromatin is an important player in checkpoint signalling.
authors: Department of Molecular Biology and Genetics, University of Aarhus, Aarhus 8000, Denmark. lbj@mb.au.dk
authors: Iben B
authors: IB
authors: Bentsen
authors: Ida
authors: I
authors: Nielsen
authors: Michael
authors: M
authors: Lisby
authors: Helena B
authors: HB
authors: Nielsen
authors: Souvik Sen
authors: SS
authors: Gupta
authors: Kamilla
authors: K
authors: Mundbjerg
authors: Anni H
authors: AH
authors: Andersen
authors: Lotte
authors: L
authors: Bjergbaek
mesh_terms: D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000378:metabolism; D064447:Checkpoint Kinase 2; D002843:Chromatin / Q000378:metabolism*; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D004275:DNA, Ribosomal / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D004706:Endodeoxyribonucleases / Q000378:metabolism* / Q000502:physiology; D005090:Exodeoxyribonucleases / Q000378:metabolism* / Q000502:physiology; D059765:Homologous Recombination; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism* / Q000502:physiology; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000378:metabolism; D056565:Sirtuin 2 / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D002843:Chromatin; D004271:DNA, Fungal; D004275:DNA, Ribosomal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C086930:RAD50 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; C086929:XRS2 protein, S cerevisiae; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C094191:MRE11 protein, S cerevisiae; C087778:SIR2 protein, S cerevisiae; D056565:Sirtuin 2
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