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Helicase/SUMO-targeted ubiquitin ligase Uls1 interacts with the Holliday junction resolvase Yen1
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.ORCID-id: 0000-0001-7721-6908
Rekke forfattare: 32019 (engelsk)Inngår i: PLOS ONE, E-ISSN 1932-6203, Vol. 14, nr 3, artikkel-id e0214102Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Resolution of branched DNA structures is pivotal for repair of stalled replication forks and meiotic recombination intermediates. The Yen1 nuclease cleaves both Holliday junctions and replication forks. We show that Yen1 interacts physically with Uls1, a suggested SUMO-targeted ubiquitin ligase that also contains a SWI/SNF-family ATPase-domain. Yen1 is SUMO-modified in its noncatalytic carboxyl terminus and DNA damage induces SUMOylation. SUMO-modification of Yen1 strengthens the interaction to Uls1, and mutations in SUMO interaction motifs in Uls1 weakens the interaction. However, Uls1 does not regulate the steady-state level of SUMO-modified Yen1 or chromatin-associated Yen1. In addition, SUMO-modification of Yen1 does not affect the catalytic activity in vitro. Consistent with a shared function for Uls1 and Yen1, mutations in both genes display similar phenotypes. Both uls1 and yen1 display negative genetic interactions with the alternative HJ-cleaving nuclease Mus81, manifested both in hypersensitivity to DNA damaging agents and in meiotic defects. Point mutations in ULS1 (uls1K975R and uls1C1330S, C1333S) predicted to inactivate the ATPase and ubiquitin ligase activities, respectively, are as defective as the null allele, indicating that both functions of Uls1 are essential. A micrococcal nuclease sequencing experiment showed that Uls1 had minimal effects on global nucleosome positioning/occupancy. Moreover, increased gene dosage of YEN1 partially alleviates the mus81 uls1 sensitivity to DNA damage. We suggest a preliminary model in which Uls1 acts in the same pathway as Yen1 to resolve branched DNA structures.

sted, utgiver, år, opplag, sider
2019. Vol. 14, nr 3, artikkel-id e0214102
HSV kategori
Identifikatorer
URN: urn:nbn:se:su:diva-168290DOI: 10.1371/journal.pone.0214102ISI: 000461889700066PubMedID: 30897139OAI: oai:DiVA.org:su-168290DiVA, id: diva2:1308034
Tilgjengelig fra: 2019-04-30 Laget: 2019-04-30 Sist oppdatert: 2022-10-13bibliografisk kontrollert
Inngår i avhandling
1. Guardians of the genome: Connecting chromatin structure and genome integrity in Saccharomyces cerevisiae
Åpne denne publikasjonen i ny fane eller vindu >>Guardians of the genome: Connecting chromatin structure and genome integrity in Saccharomyces cerevisiae
2022 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Branched DNA structures arise during recombination and during other DNA repair processes such as repair of stalled replication forks. One branched structure, the Holliday Junction (HJ), is a four-way junction that can connect sister chromatids or homologous chromosomes. We show a physical interaction between the HJ-cleaving enzyme Yen1 and Uls1. Uls1 shares homology with the SWI/SNF-family of chromatin remodelers and has a SUMO-targeted ubiquitin ligase activity, but its cellular role remains poorly characterized. SUMOylation of Yen1 strengthened the interaction with Uls1 and both yen1 and uls1 displayed negative genetic interactions with mutations in a gene encoding an alternative HJ-cleaving enzyme MUS81. Hence, Uls1, Yen1 and Mus81 appeared to be partially redundant. A uls1 strain, however, showed minimal changes in nucleosome positioning and occupancy, as determined by a genome-wide micrococcal nuclease sequencing (MNase-Seq) analysis. Together, these results suggested a role of Uls1 in Yen1-mediated HJ resolution rather than SWI/SNF-like chromatin remodeling activity.

Telomeres protect the ends of the linear chromosomes in eukaryotes from both degradation and mistaken recognition as DNA damage. In Saccharomyces cerevisiae, silent information regulator (Sir) proteins silence transcription from telomeres, by forming a higher-order chromatin structure reminiscent of heterochromatin. An MNase-Seq analysis comparing nucleosome occupancy/positioning in a sir2 mutant with a WT strain showed that the histone deacetylase Sir2 was required for stabilizing a nucleosome in subtelomeric X-elements. The Reb1 transcription factor destabilized the same nucleosome, hence competing with Sir2. Transcription at telomeres results in a species of long noncoding RNA called telomeric repeat containing RNAs (TERRAs). Sir2 and Reb1 limited TERRA steady-state levels, by different mechanisms where Reb1 presumably blocked transcription elongation. The transcriptional start sites of TERRAs mapped just centromere proximal to the Sir2-stabilized nucleosome and the nucleosome occupancy of X-elements was dependent on the position close to the telomeres rather than the DNA sequence of the X-element itself.

Further analysis of nucleosome occupancy in the sir2 mutant strain revealed Sir2-stabilized nucleosomes in the intergenic spacer region (IGS) of the ribosomal DNA (rDNA) locus. The lowered nucleosome occupancy in the IGS overlapped with the promoter for a non-coding RNA, which in turn regulates rDNA copy number. The sir2 mutant also displayed indirect effects on nucleosome occupancy at cell-type specific genes.

Together, the results reveal important roles for Sir2 in regulating nucleosome occupancy/positioning and begin exploring links between chromatin structure, TERRA transcription and maintenance of genome stability.

sted, utgiver, år, opplag, sider
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 2022. s. 41
HSV kategori
Forskningsprogram
molekylär biovetenskap
Identifikatorer
urn:nbn:se:su:diva-209904 (URN)978-91-8014-058-4 (ISBN)978-91-8014-059-1 (ISBN)
Disputas
2022-11-29, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2022-11-03 Laget: 2022-10-13 Sist oppdatert: 2022-11-21bibliografisk kontrollert

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