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

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  • Iben Bach Bentsen, Denmark
  • Ida Nielsen, Denmark
  • Michael Lisby, University of Copenhagen, Denmark
  • Helena Berner Nedergaard Nielsen
  • ,
  • Souvik Sen Gupta, Denmark
  • Kamilla Mundbjerg, Denmark
  • Anni Hangaard Andersen
  • Lotte Bjergbæk
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 co
Original languageEnglish
JournalNucleic Acids Research
Volume41
Issue5
Pages (from-to)3173-3189
Number of pages17
ISSN0305-1048
DOIs
Publication statusPublished - 1 Feb 2013

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