MRX protects fork integrity at protein-DNA barriers, and its absence causes checkpoint activation dependent on chromatin context

• Published in: Nucleic acids research 2013-03, Vol.41 (5), p.3173-3189
• Main Authors: Bentsen, Iben B, Nielsen, Ida, Lisby, Michael, Nielsen, Helena B, Gupta, Souvik Sen, Mundbjerg, Kamilla, Andersen, Anni H, Bjergbaek, Lotte
• Format: Article
• Language: English
• Subjects: Cell Cycle Checkpoints; Cell Cycle Proteins - metabolism; Checkpoint Kinase 2; Chromatin - metabolism; DNA Replication; DNA, Fungal - genetics; DNA, Fungal - metabolism; DNA, Ribosomal - genetics; DNA, Ribosomal - metabolism; DNA, Single-Stranded - metabolism; DNA-Binding Proteins - metabolism; DNA-Binding Proteins - physiology; Endodeoxyribonucleases - metabolism; Endodeoxyribonucleases - physiology; Exodeoxyribonucleases - metabolism; Exodeoxyribonucleases - physiology; Genome Integrity, Repair and; Homologous Recombination; Protein-Serine-Threonine Kinases - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae Proteins - metabolism; Saccharomyces cerevisiae Proteins - physiology; Silent Information Regulator Proteins, Saccharomyces cerevisiae - metabolism; Sirtuin 2 - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkt051

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.