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.

• Klíčová slova
• DNA, Genomic instability,
• MeSH
• DNA fungální genetika   metabolismus   MeSH
• DNA-helikasy chemie   genetika   metabolismus   MeSH
• fosforylace MeSH
• kinetika MeSH
• metabolické sítě a dráhy MeSH
• poškození DNA MeSH
• proteinové domény MeSH
• rekombinantní fúzní proteiny chemie   genetika   metabolismus   MeSH
• replikace DNA MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae enzymologie   genetika   růst a vývoj   MeSH
• stabilita enzymů MeSH
• sumoylace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA fungální MeSH
• DNA-helikasy MeSH
• DNA2 protein, S cerevisiae MeSH Prohlížeč
• rekombinantní fúzní proteiny MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• Siz2 protein, S cerevisiae MeSH Prohlížeč

Protein modifications regulate both DNA repair levels and pathway choice. How each modification achieves regulatory effects and how different modifications collaborate with each other are important questions to be answered. Here, we show that sumoylation regulates double-strand break repair partly by modifying the end resection factor Sae2. This modification is conserved from yeast to humans, and is induced by DNA damage. We mapped the sumoylation site of Sae2 to a single lysine in its self-association domain. Abolishing Sae2 sumoylation by mutating this lysine to arginine impaired Sae2 function in the processing and repair of multiple types of DNA breaks. We found that Sae2 sumoylation occurs independently of its phosphorylation, and the two modifications act in synergy to increase soluble forms of Sae2. We also provide evidence that sumoylation of the Sae2-binding nuclease, the Mre11-Rad50-Xrs2 complex, further increases end resection. These findings reveal a novel role for sumoylation in DNA repair by regulating the solubility of an end resection factor. They also show that collaboration between different modifications and among multiple substrates leads to a stronger biological effect.

• MeSH
• DNA vazebné proteiny genetika   MeSH
• dvouřetězcové zlomy DNA MeSH
• endodeoxyribonukleasy genetika   MeSH
• endonukleasy genetika   MeSH
• exodeoxyribonukleasy genetika   MeSH
• fosforylace MeSH
• lidé MeSH
• oprava DNA spojením konců genetika   MeSH
• oprava DNA genetika   MeSH
• poškození DNA genetika   MeSH
• rozpustnost MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• Saccharomyces cerevisiae MeSH
• sumoylace genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• endodeoxyribonukleasy MeSH
• endonukleasy MeSH
• exodeoxyribonukleasy MeSH
• MRE11 protein, S cerevisiae MeSH Prohlížeč
• RAD50 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• SAE2 protein, S cerevisiae MeSH Prohlížeč
• XRS2 protein, S cerevisiae MeSH Prohlížeč

The 5'-3' resection of DNA ends is a prerequisite for the repair of DNA double strand breaks by homologous recombination, microhomology-mediated end joining, and single strand annealing. Recent studies in yeast have shown that, following initial DNA end processing by the Mre11-Rad50-Xrs2 complex and Sae2, the extension of resection tracts is mediated either by exonuclease 1 or by combined activities of the RecQ family DNA helicase Sgs1 and the helicase/endonuclease Dna2. Although human DNA2 has been shown to cooperate with the BLM helicase to catalyze the resection of DNA ends, it remains a matter of debate whether another human RecQ helicase, WRN, can substitute for BLM in DNA2-catalyzed resection. Here we present evidence that WRN and BLM act epistatically with DNA2 to promote the long-range resection of double strand break ends in human cells. Our biochemical experiments show that WRN and DNA2 interact physically and coordinate their enzymatic activities to mediate 5'-3' DNA end resection in a reaction dependent on RPA. In addition, we present in vitro and in vivo data suggesting that BLM promotes DNA end resection as part of the BLM-TOPOIIIα-RMI1-RMI2 complex. Our study provides new mechanistic insights into the process of DNA end resection in mammalian cells.

• Klíčová slova
• DNA Damage, DNA Helicase, DNA Recombination, DNA Repair, Genomic Instability, RecQ,
• MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• DNA-helikasy genetika   metabolismus   MeSH
• DNA genetika   metabolismus   MeSH
• dvouřetězcové zlomy DNA * MeSH
• enzymy opravy DNA genetika   metabolismus   MeSH
• exodeoxyribonukleasy genetika   metabolismus   MeSH
• genetická epistáze fyziologie   MeSH
• HEK293 buňky MeSH
• helikasy RecQ genetika   metabolismus   MeSH
• helikáza Wernerova syndromu MeSH
• homologní protein MRE11 MeSH
• hydrolasy působící na anhydridy kyselin MeSH
• lidé MeSH
• multienzymové komplexy genetika   metabolismus   MeSH
• ubikvitin aktivující enzymy genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Bloom syndrome protein MeSH Prohlížeč
• DNA vazebné proteiny MeSH
• DNA-helikasy MeSH
• DNA MeSH
• DNA2 protein, human MeSH Prohlížeč
• enzymy opravy DNA MeSH
• exodeoxyribonukleasy MeSH
• helikasy RecQ MeSH
• helikáza Wernerova syndromu MeSH
• homologní protein MRE11 MeSH
• hydrolasy působící na anhydridy kyselin MeSH
• MRE11 protein, human MeSH Prohlížeč
• multienzymové komplexy MeSH
• RAD50 protein, human MeSH Prohlížeč
• UBA2 protein, human MeSH Prohlížeč
• ubikvitin aktivující enzymy MeSH
• WRN protein, human MeSH Prohlížeč

While the mechanisms governing DNA damage response and repair are fundamentally conserved, cross-kingdom comparisons indicate that they differ in many aspects due to differences in life-styles and developmental strategies. In photosynthetic organisms these differences have not been fully explored because gene-discovery approaches are mainly based on homology searches with known DDR/DNA repair proteins. Here we performed a forward genetic screen in the green algae Chlamydomonas reinhardtii to identify genes deficient in DDR/DNA repair. We isolated five insertional mutants that were sensitive to various genotoxic insults and two of them exhibited altered efficiency of transgene integration. To identify genomic regions disrupted in these mutants, we established a novel adaptor-ligation strategy for the efficient recovery of the insertion flanking sites. Four mutants harbored deletions that involved known DNA repair factors, DNA Pol zeta, DNA Pol theta, SAE2/COM1, and two neighbouring genes encoding ERCC1 and RAD17. Deletion in the last mutant spanned two Chlamydomonas-specific genes with unknown function, demonstrating the utility of this approach for discovering novel factors involved in genome maintenance.

• MeSH
• bakteriální transformace účinky léků   MeSH
• Chlamydomonas reinhardtii účinky léků   genetika   MeSH
• genetické vektory genetika   MeSH
• hydroxymočovina farmakologie   toxicita   MeSH
• inzerční mutageneze * MeSH
• mutace MeSH
• oprava DNA * MeSH
• pořadí genů genetika   MeSH
• poškození DNA * účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hydroxymočovina MeSH