Abstract
Structure-specific endonucleases contribute to the maintenance of genome integrity by cleaving DNA intermediates that need to be resolved for faithful DNA repair, replication, or recombination. Despite advances in the understanding of their function and regulation, it is less clear how these proteins respond to genotoxic stress. Here, we show that the structure-specific endonuclease Mus81-Mms4/EME1 relocalizes to subnuclear foci following DNA damage and colocalizes with the endonucleases Rad1-Rad10 (XPF-ERCC1) and Slx1-Slx4. Recruitment takes place into a class of stress foci defined by Cmr1/WDR76, a protein involved in preserving genome stability, and depends on the E2-ubiquitin-conjugating enzyme Rad6 and the E3-ubiquitin ligase Bre1. Foci dynamics show that, in the presence of DNA intermediates that need resolution by Mus81-Mms4, Mus81 foci persist until this endonuclease is activated by Mms4 phosphorylation. Our data suggest that subnuclear relocalization is relevant for the function of Mus81-Mms4 and, probably, of the endonucleases that colocalize with it.
Highlights
The function of structure-specific endonucleases is important for cell survival under DNA damage conditions, which can be caused by endogenous and environmental agents, as well as by cancer therapeutic treatments
Mus81-Mms4 Relocalizes to Subnuclear Foci in Response to DNA Damage To study the localization of different structure-specific endonucleases, we tagged the proteins with GFP, which yielded Saccharomyces cerevisiae strains that behaved to parental controls (Figure S1A)
We started analyzing Mus81Mms4 and found that in untreated cells, the two subunits appeared diffused in the nucleus, whereas they formed subnuclear foci after treatment with the DNA-damaging agent methyl methanesulfonate (MMS) (Figures 1A and 1F)
Summary
The function of structure-specific endonucleases is important for cell survival under DNA damage conditions, which can be caused by endogenous and environmental agents, as well as by cancer therapeutic treatments. Protein relocalization is a hallmark of the cellular response to DNA damage or replicative stress (Lisby et al, 2004; Tkach et al, 2012), but the potential regulation of structure-specific endonucleases by this mechanism remains poorly understood. We show that Mus81-Mms4/EME1 and other endonucleases undergo cellular relocalization upon DNA damage. Mus81-Mms is recruited into subnuclear foci following genotoxic stress and colocalizes at these sites with the endonucleases Rad1-Rad and Slx1Slx. Mus81-Mms is recruited into subnuclear foci following genotoxic stress and colocalizes at these sites with the endonucleases Rad1-Rad and Slx1Slx4 This relocalization could be important for the function of these proteins
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