Dna2 is an essential nuclease-helicase that acts in several distinct DNA metabolic pathways including DNA replication and recombination. To balance these functions and prevent unscheduled DNA degradation, Dna2 activities must be regulated. Here we show that Saccharomyces cerevisiae Dna2 function is controlled by sumoylation. We map the sumoylation sites to the N-terminal regulatory domain of Dna2 and show that in vitro sumoylation of recombinant Dna2 impairs its nuclease but not helicase activity. In cells, the total levels of the non-sumoylatable Dna2 variant are elevated. However, non-sumoylatable Dna2 shows impaired nuclear localization and reduced recruitment to foci upon DNA damage. Non-sumoylatable Dna2 reduces the rate of DNA end resection, as well as impedes cell growth and cell cycle progression through S phase. Taken together, these findings show that in addition to Dna2 phosphorylation described previously, Dna2 sumoylation is required for the homeostasis of the Dna2 protein function to promote genome stability.

Department of Biology Masaryk University Kamenice 5 625 00 Brno Czech Republic 4International Clinical Research Center St Anne's University Hospital 656 91 Brno Czech Republic

Department of Biology Masaryk University Kamenice 5 625 00 Brno Czech Republic 4International Clinical Research Center St Anne's University Hospital 656 91 Brno Czech Republic 5National Center for Biomolecular Research Masaryk University Kamenice 5 625 00 Brno Czech Republic

Institute for Research in Biomedicine Università della Svizzera italiana 8093 Zürich Switzerland

Institute for Research in Biomedicine Università della Svizzera italiana Faculty of Biomedical Sciences Via Vincenzo Vela 6 6500 Bellinzona Switzerland

Institute of Molecular Cancer Research University of Zürich Winterthurerstrasse 190 8057 Zürich Switzerland

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