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 Institute of Biochemistry Eidgenössische Technische Hochschule 8093 Zürich Switzerland

Department of Biology Masaryk University Kamenice 5 625 00 Brno Czech Republic

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

International Clinical Research Center St Anne's University Hospital 656 91 Brno Czech Republic

National Center for Biomolecular Research Masaryk University Kamenice 5 625 00 Brno Czech Republic

Zobrazit více v PubMed

Jackson SP, Durocher D. Regulation of DNA damage responses by ubiquitin and SUMO. Mol. Cell. 2013;49:795–807. doi: 10.1016/j.molcel.2013.01.017. PubMed DOI

Jentsch S, Psakhye I. Control of nuclear activities by substrate-selective and protein-group SUMOylation. Annu. Rev. Genet. 2013;47:167–186. doi: 10.1146/annurev-genet-111212-133453. PubMed DOI

Ulrich HD. Two-way communications between ubiquitin-like modifiers and DNA. Nat. Struct. Mol. Biol. 2014;21:317–324. doi: 10.1038/nsmb.2805. PubMed DOI

Altmannova V, Kolesar P, Krejci L. SUMO wrestles with recombination. Biomolecules. 2012;2:350–375. doi: 10.3390/biom2030350. PubMed DOI PMC

Bergink S, Jentsch S. Principles of ubiquitin and SUMO modifications in DNA repair. Nature. 2009;458:461–467. doi: 10.1038/nature07963. PubMed DOI

Liebelt F, Vertegaal AC. Ubiquitin-dependent and independent roles of SUMO in proteostasis. Am. J. Physiol. Cell Physiol. 2016;311:C284–C296. doi: 10.1152/ajpcell.00091.2016. PubMed DOI PMC

Sarangi P, Zhao X. SUMO-mediated regulation of DNA damage repair and responses. Trends Biochem. Sci. 2015;40:233–242. doi: 10.1016/j.tibs.2015.02.006. PubMed DOI PMC

Kerscher O. SUMO junction-what’s your function? New insights through SUMO-interacting motifs. EMBO Rep. 2007;8:550–555. doi: 10.1038/sj.embor.7400980. PubMed DOI PMC

Psakhye I, Jentsch S. Protein group modification and synergy in the SUMO pathway as exemplified in DNA repair. Cell. 2012;151:807–820. doi: 10.1016/j.cell.2012.10.021. PubMed DOI

Budd ME, Choe W, Campbell JL. The nuclease activity of the yeast DNA2 protein, which is related to the RecB-like nucleases, is essential in vivo. J. Biol. Chem. 2000;275:16518–16529. doi: 10.1074/jbc.M909511199. PubMed DOI

Formosa T, Nittis T. Dna2 mutants reveal interactions with Dna polymerase alpha and Ctf4, a Pol alpha accessory factor, and show that full Dna2 helicase activity is not essential for growth. Genetics. 1999;151:1459–1470. PubMed PMC

Bae SH, Bae KH, Kim JA, Seo YS. RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes. Nature. 2001;412:456–461. doi: 10.1038/35086609. PubMed DOI

Budd ME, Choe WC, Campbell JL. DNA2 encodes a DNA helicase essential for replication of eukaryotic chromosomes. J. Biol. Chem. 1995;270:26766–26769. doi: 10.1074/jbc.270.45.26766. PubMed DOI

Cejka P, et al. DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2. Nature. 2010;467:112–116. doi: 10.1038/nature09355. PubMed DOI PMC

Kumar S, Burgers PM. Lagging strand maturation factor Dna2 is a component of the replication checkpoint initiation machinery. Genes Dev. 2013;27:313–321. doi: 10.1101/gad.204750.112. PubMed DOI PMC

Olmezer G, et al. Replication intermediates that escape Dna2 activity are processed by Holliday junction resolvase Yen1. Nat. Commun. 2016;7:13157. doi: 10.1038/ncomms13157. PubMed DOI PMC

Thangavel S, et al. DNA2 drives processing and restart of reversed replication forks in human cells. J. Cell Biol. 2015;208:545–562. doi: 10.1083/jcb.201406100. PubMed DOI PMC

Hu J, et al. The intra-S phase checkpoint targets Dna2 to prevent stalled replication forks from reversing. Cell. 2012;149:1221–1232. doi: 10.1016/j.cell.2012.04.030. PubMed DOI

Zhu Z, Chung WH, Shim EY, Lee SE, Ira G. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell. 2008;134:981–994. doi: 10.1016/j.cell.2008.08.037. PubMed DOI PMC

Levikova M, Pinto C, Cejka P. The motor activity of DNA2 functions as an ssDNA translocase to promote DNA end resection. Genes Dev. 2017;31:493–502. doi: 10.1101/gad.295196.116. PubMed DOI PMC

Niu H, et al. Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae. Nature. 2010;467:108–111. doi: 10.1038/nature09318. PubMed DOI PMC

Miller AS, et al. A novel role of the Dna2 translocase function in DNA break resection. Genes Dev. 2017;31:503–510. doi: 10.1101/gad.295659.116. PubMed DOI PMC

Cannavo E, Cejka P, Kowalczykowski SC. Relationship of DNA degradation by Saccharomyces cerevisiae exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection. Proc. Natl Acad. Sci. USA. 2013;110:E1661–E1668. doi: 10.1073/pnas.1305166110. PubMed DOI PMC

Nicolette ML, et al. Mre11-Rad50-Xrs2 and Sae2 promote 5’ strand resection of DNA double-strand breaks. Nat. Struct. Mol. Biol. 2010;17:1478–1485. doi: 10.1038/nsmb.1957. PubMed DOI PMC

Chen X, et al. Cell cycle regulation of DNA double-strand break end resection by Cdk1-dependent Dna2 phosphorylation. Nat. Struct. Mol. Biol. 2011;18:1015–1019. doi: 10.1038/nsmb.2105. PubMed DOI PMC

Nimonkar AV, Ozsoy AZ, Genschel J, Modrich P, Kowalczykowski SC. Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair. Proc. Natl Acad. Sci. USA. 2008;105:16906–16911. doi: 10.1073/pnas.0809380105. PubMed DOI PMC

Sarangi P, et al. Sumoylation influences DNA break repair partly by increasing the solubility of a conserved end resection protein. PLoS Genet. 2015;11:e1004899. doi: 10.1371/journal.pgen.1004899. PubMed DOI PMC

Chen YJ, et al. S. cerevisiae Mre11 recruits conjugated SUMO moieties to facilitate the assembly and function of the Mre11-Rad50-Xrs2 complex. Nucl. Acids Res. 2016;44:2199–2213. doi: 10.1093/nar/gkv1523. PubMed DOI PMC

Kolesar P, Sarangi P, Altmannova V, Zhao X, Krejci L. Dual roles of the SUMO-interacting motif in the regulation of Srs2 sumoylation. Nucl. Acids Res. 2012;40:7831–7843. doi: 10.1093/nar/gks484. PubMed DOI PMC

Bologna Serena, Altmannova Veronika, Valtorta Emanuele, Koenig Christiane, Liberali Prisca, Gentili Christian, Anrather Dorothea, Ammerer Gustav, Pelkmans Lucas, Krejci Lumir, Ferrari Stefano. Sumoylation regulates EXO1 stability and processing of DNA damage. Cell Cycle. 2015;14(15):2439–2450. doi: 10.1080/15384101.2015.1060381. PubMed DOI PMC

Ulrich HD, Davies AA. In vivo detection and characterization of sumoylation targets in Saccharomyces cerevisiae. Methods Mol. Biol. 2009;497:81–103. doi: 10.1007/978-1-59745-566-4_6. PubMed DOI

Davies AA, Ulrich HD. Detection of PCNA modifications in Saccharomyces cerevisiae. Methods Mol. Biol. 2012;920:543–567. doi: 10.1007/978-1-61779-998-3_36. PubMed DOI

Tsabar Michael, Eapen Vinay V., Mason Jennifer M., Memisoglu Gonen, Waterman David P., Long Marcus J., Bishop Douglas K., Haber James E. Caffeine impairs resection during DNA break repair by reducing the levels of nucleases Sae2 and Dna2. Nucleic Acids Research. 2015;43(14):6889–6901. doi: 10.1093/nar/gkv520. PubMed DOI PMC

Bermudez-Lopez M, Aragon L. Smc5/6 complex regulates Sgs1 recombination functions. Curr. Genet. 2017;63:381–388. doi: 10.1007/s00294-016-0648-5. PubMed DOI PMC

Hoege C, Pfander B, Moldovan GL, Pyrowolakis G, Jentsch S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature. 2002;419:135–141. doi: 10.1038/nature00991. PubMed DOI

Saponaro M, et al. Cdk1 targets Srs2 to complete synthesis-dependent strand annealing and to promote recombinational repair. PLoS Genet. 2010;6:e1000858. doi: 10.1371/journal.pgen.1000858. PubMed DOI PMC

Bae KH, et al. Bimodal interaction between replication-protein A and Dna2 is critical for Dna2 function both in vivo and in vitro. Nucl. acids Res. 2003;31:3006–3015. doi: 10.1093/nar/gkg422. PubMed DOI PMC

Balakrishnan L, Polaczek P, Pokharel S, Campbell JL, Bambara RA. Dna2 exhibits a unique strand end-dependent helicase function. J. Biol. Chem. 2010;285:38861–38868. doi: 10.1074/jbc.M110.165191. PubMed DOI PMC

Levikova M, Klaue D, Seidel R, Cejka P. Nuclease activity of Saccharomyces cerevisiae Dna2 inhibits its potent DNA helicase activity. Proc. Natl Acad. Sci. USA. 2013;110:E1992–E2001. doi: 10.1073/pnas.1300390110. PubMed DOI PMC

Lee CH, et al. The N-terminal 45-kDa domain of Dna2 endonuclease/helicase targets the enzyme to secondary structure DNA. J. Biol. Chem. 2013;288:9468–9481. doi: 10.1074/jbc.M112.418715. PubMed DOI PMC

Bae SH, et al. Tripartite structure of Saccharomyces cerevisiae Dna2 helicase/endonuclease. Nucl. Acids Res. 2001;29:3069–3079. doi: 10.1093/nar/29.14.3069. PubMed DOI PMC

Widlund PO, Davis TN. A high-efficiency method to replace essential genes with mutant alleles in yeast. Yeast. 2005;22:769–774. doi: 10.1002/yea.1244. PubMed DOI PMC

White CI, Haber JE. Intermediates of recombination during mating type switching in Saccharomyces cerevisiae. Embo J. 1990;9:663–673. doi: 10.1002/j.1460-2075.1990.tb08158.x. PubMed DOI PMC

Budd ME, Reis CC, Smith S, Myung K, Campbell JL. Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase delta. Mol. Cell. Biol. 2006;26:2490–2500. doi: 10.1128/MCB.26.7.2490-2500.2006. PubMed DOI PMC

Zhou J, Monson EK, Teng SC, Schulz VP, Zakian VA. Pif1p helicase, a catalytic inhibitor of telomerase in yeast. Science. 2000;289:771–774. doi: 10.1126/science.289.5480.771. PubMed DOI

Parenteau J, Wellinger RJ. Accumulation of single-stranded DNA and destabilization of telomeric repeats in yeast mutant strains carrying a deletion of RAD27. Mol. Cell. Biol. 1999;19:4143–4152. doi: 10.1128/MCB.19.6.4143. PubMed DOI PMC

Miteva M, Keusekotten K, Hofmann K, Praefcke GJ, Dohmen RJ. Sumoylation as a signal for polyubiquitylation and proteasomal degradation. Subcell. Biochem. 2010;54:195–214. doi: 10.1007/978-1-4419-6676-6_16. PubMed DOI

Guo Z, et al. Sequential posttranslational modifications program FEN1 degradation during cell-cycle progression. Mol. Cell. 2012;47:444–456. doi: 10.1016/j.molcel.2012.05.042. PubMed DOI PMC

Gibbs-Seymour I, et al. Ubiquitin-SUMO circuitry controls activated fanconi anemia ID complex dosage in response to DNA damage. Mol. Cell. 2015;57:150–164. doi: 10.1016/j.molcel.2014.12.001. PubMed DOI PMC

Ayyagari R, Gomes XV, Gordenin DA, Burgers PM. Okazaki fragment maturation in yeast. I. Distribution of functions between FEN1 AND DNA2. J. Biol. Chem. 2003;278:1618–1625. doi: 10.1074/jbc.M209801200. PubMed DOI

Levikova M, Cejka P. The Saccharomyces cerevisiae Dna2 can function as a sole nuclease in the processing of Okazaki fragments in DNA replication. Nucl. Acids Res. 2015;43:7888–7897. doi: 10.1093/nar/gkv710. PubMed DOI PMC

Fiorentino DF, Crabtree GR. Characterization of Saccharomyces cerevisiae dna2 mutants suggests a role for the helicase late in S phase. Mol. Biol. cell. 1997;8:2519-2537. doi: 10.1091/mbc.8.12.2519. PubMed DOI PMC

Duxin JP, et al. Human Dna2 is a nuclear and mitochondrial DNA maintenance protein. Mol. Cell. Biol. 2009;29:4274–4282. doi: 10.1128/MCB.01834-08. PubMed DOI PMC

Vallen EA, Cross FR. Mutations in RAD27 define a potential link between G1 cyclins and DNA replication. Mol. Cell. Biol. 1995;15:4291–4302. doi: 10.1128/MCB.15.8.4291. PubMed DOI PMC

Kumar S, et al. Inhibition of DNA2 nuclease as a therapeutic strategy targeting replication stress in cancer cells. Oncogenesis. 2017;6:e319. doi: 10.1038/oncsis.2017.15. PubMed DOI PMC

Peng G, et al. Human nuclease/helicase DNA2 alleviates replication stress by promoting DNA end resection. Cancer Res. 2012;72:2802–2813. doi: 10.1158/0008-5472.CAN-11-3152. PubMed DOI PMC

Anand R, Pinto C, Cejka P. Methods to Study DNA End Resection I: Recombinant Protein Purification. Methods Enzym. 2018;600:25–66. doi: 10.1016/bs.mie.2017.11.008. PubMed DOI

Altmannova V, et al. Rad52 SUMOylation affects the efficiency of the DNA repair. Nucl. Acids Res. 2010;38:4708–4721. doi: 10.1093/nar/gkq195. PubMed DOI PMC

Takahashi Y, Toh EA, Kikuchi Y. Comparative analysis of yeast PIAS-type SUMO ligases in vivo and in vitro. J. Biochem. 2003;133:415–422. doi: 10.1093/jb/mvg054. PubMed DOI

Iaccarino I, et al. MSH6, a Saccharomyces cerevisiae protein that binds to mismatches as a heterodimer with MSH2. Curr. Biol. 1996;6:484–486. doi: 10.1016/S0960-9822(02)00516-X. PubMed DOI

Sarangi P, et al. Sumoylation of the Rad1 nuclease promotes DNA repair and regulates its DNA association. Nucl. Acids Res. 2014;42:6393–6404. doi: 10.1093/nar/gku300. PubMed DOI PMC

Cejka P, Kowalczykowski SC. The full-length Saccharomyces cerevisiae Sgs1 protein is a vigorous DNA helicase that preferentially unwinds holliday junctions. J. Biol. Chem. 2010;285:8290–8301. doi: 10.1074/jbc.M109.083196. PubMed DOI PMC

Pinto C, Anand R, Cejka P. Methods to study DNA end resection II: Biochemical reconstitution assays. Methods Enzym. 2018;600:67–106. doi: 10.1016/bs.mie.2017.11.009. PubMed DOI

Kowalczykowski SC, Krupp RA. Effects of Escherichia coli SSB protein on the single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein. Evidence that SSB protein facilitates the binding of RecA protein to regions of secondary structure within single-stranded DNA. J. Mol. Biol. 1987;193:97–113. doi: 10.1016/0022-2836(87)90630-9. PubMed DOI

Janke C, et al. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast. 2004;21:947–962. doi: 10.1002/yea.1142. PubMed DOI

Liu C, Apodaca J, Davis LE, Rao H. Proteasome inhibition in wild-type yeast Saccharomyces cerevisiae cells. Biotechniques. 2007;42:156-+. doi: 10.2144/000112389. PubMed DOI

Giannattasio M, et al. Exo1 competes with repair synthesis, converts NER intermediates to long ssDNA gaps, and promotes checkpoint activation. Mol. Cell. 2010;40:50–62. doi: 10.1016/j.molcel.2010.09.004. PubMed DOI

Silva S, Gallina I, Eckert-Boulet N, Lisby M. Live cell microscopy of DNA damage response in Saccharomyces cerevisiae. Methods Mol. Biol. 2012;920:433–443. doi: 10.1007/978-1-61779-998-3_30. PubMed DOI