Reversible Top1 cleavage complexes are stabilized strand-specifically at the ribosomal replication fork barrier and contribute to ribosomal DNA stability

Krawczyk, Claudia; Dion, Vincent; Schär, Primo; Fritsch, Olivier

doi:10.1093/nar/gku148

Cited by 23 publications

(39 citation statements)

References 54 publications

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“…Previously, Slx5 and Sir2 were found to interact physically based on yeast two-hybrid and Figure 8 Hierarchical protein binding at the rDNA locus and intersections with SUMO. Fob1 binds to the replication fork barrier (RFB) and is responsible for localizing Net1, Tof2, and Top1 to this site (Huang et al 2006;Krawczyk et al 2014). Tof2 increases the interaction of Fob1 with the rDNA (thin arrow).…”

Section: Discussionmentioning

confidence: 99%

“…Krawczyk et al 2014). Therefore, the regulation/ binding of Fob1 can have a large impact on multiple rDNAbinding proteins.…”

mentioning

confidence: 99%

“…Proper Tof2 localization is required for the proper placement of the cohibin complex (Huang et al 2006), which ultimately is needed for cohesin and condensin localization (Johzuka and Horiuchi 2009). The combination of Tof2 and Fob1 is required for DNA helicases and topoisomerases to bind to the rDNA (Weitao et al 2003;Krawczyk et al 2014). Therefore, the regulation/ binding of Fob1 can have a large impact on multiple rDNAbinding proteins.…”

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confidence: 99%

“…Paradoxically, Fob1 both is required for and acts to prevent homologous rDNA recombination (Kobayashi et al 1998). Stalled replication forks create torsional strain in the replicating DNA, and Fob1 recruits topoisomerase I and DNA helicases to the stalled fork to relieve the strain (Weitao et al 2003;Krawczyk et al 2014). Conversely, the RFB is downstream of the 35S transcription region and prevents the DNA polymerase machinery from proceeding into the RNAPItranscribed region.…”

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confidence: 99%

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SUMO Pathway Modulation of Regulatory Protein Binding at the Ribosomal DNA Locus inSaccharomyces cerevisiae

Gillies

Hickey

et al. 2016

Genetics

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In this report, we identify cellular targets of Ulp2, one of two Saccharomyces cerevisiae small ubiquitin-related modifier (SUMO) proteases, and investigate the function of SUMO modification of these proteins. PolySUMO conjugates from ulp2D and ulp2D slx5D cells were isolated using an engineered affinity reagent containing the four SUMO-interacting motifs (SIMs) of Slx5, a component of the Slx5/Slx8 SUMO-targeted ubiquitin ligase (STUbL). Two proteins identified, Net1 and Tof2, regulate ribosomal DNA (rDNA) silencing and were found to be hypersumoylated in ulp2D, slx5D, and ulp2D slx5D cells. The increase in sumoylation of Net1 and Tof2 in ulp2D, but not ulp1ts cells, indicates that these nucleolar proteins are specific substrates of Ulp2. Based on quantitative chromatin-immunoprecipitation assays, both Net1 and Tof2 lose binding to their rDNA sites in ulp2D cells and both factors largely regain this association in ulp2D slx5D. A parsimonious interpretation of these results is that hypersumoylation of these proteins causes them to be ubiquitylated by Slx5/ Slx8, impairing their association with rDNA. Fob1, a protein that anchors both Net1 and Tof2 to the replication-fork barrier (RFB) in the rDNA repeats, is sumoylated in wild-type cells, and its modification levels increase specifically in ulp2D cells. Fob1 experiences a 50% reduction in rDNA binding in ulp2D cells, which is also rescued by elimination of Slx5. Additionally, overexpression of Sir2, another RFBassociated factor, suppresses the growth defect of ulp2D cells. Our data suggest that regulation of rDNA regulatory proteins by Ulp2 and the Slx5/Slx8 STUbL may be the cause of multiple ulp2D cellular defects. KEYWORDS SUMO; Ulp2; rDNA; RENT complex; Fob1 P OST-TRANSLATIONAL protein modifications provide a common mechanism for regulating protein-protein interactions, protein localization, and protein degradation. The small ubiquitin-related modifier (SUMO) protein family modifies target proteins through covalent attachment to lysine side chains (Johnson 2004). The function of substrate "sumoylation" varies with the protein that is sumoylated, but a common role is to modulate the assembly of large protein complexes either by creating additional binding sites or by blocking existing sites (Kerscher 2007).Protein sumoylation occurs through an enzymatic cascade similar to ubiquitylation (Johnson 2004). In Saccharomyces cerevisiae, a single gene, SMT3, codes for SUMO, and as in most species, SUMO ligation is essential for viability. Removal of SUMO from target proteins is also an important regulatory step. S. cerevisiae has two SUMO proteases, Ulp1 and Ulp2 (Hickey et al. 2012). Most Ulp1 is bound to the nuclear pore complex (NPC), and the enzyme is essential for cell-cycle progression. NPC tethering regulates Ulp1 activity by restricting its access to sumoylated proteins (Li and Hochstrasser 2003;Panse et al. 2003). Ulp1 also processes the SUMO precursor by removing its last three residues, thereby exposing the C-terminal Gly-Gly motif required for SU...

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Section: Discussionmentioning

confidence: 99%

“…Krawczyk et al 2014). Therefore, the regulation/ binding of Fob1 can have a large impact on multiple rDNAbinding proteins.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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SUMO Pathway Modulation of Regulatory Protein Binding at the Ribosomal DNA Locus inSaccharomyces cerevisiae

Gillies

Hickey

et al. 2016

Genetics

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“…Top1 cleavage complexes accumulate at damaged DNA sites (8,35) under normal conditions (22,36), and TDP1 repairs not only Top1-mediated DNA damage but also endogenous oxidative DNA lesions (2,8,12). Both of these types of damage can result in increased mortality, and we therefore tested whether TDP1 influences the lifespan of tdp1Δ flies.…”

Section: Tdp1 Mutant Females Have Short Lifespans and Defective Climbingmentioning

confidence: 99%

Neuroprotection and repair of 3′-blocking DNA ends by glaikit (gkt) encoding Drosophila tyrosyl-DNA phosphodiesterase 1 (TDP1)

Guo¹,

Dexheimer

Pommier

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Tyrosyl-DNA phosphodiesterase (TDP1) is a phylogenetically conserved enzyme critical for the removal of blocking lesions at the 3′ ends of DNA or RNA. This study analyzes the Drosophila TDP1 gene ortholog glaikit (gkt) and its possible role(s) in the repair of endogenous DNA lesions and neuroprotection. To do so, we studied a homozygous PiggyBac insertion (c03958) that disrupts the 5′ UTR of gkt. Protein extracts of c03958 flies were defective in hydrolyzing 3′-DNA-tyrosyl residues, demonstrating that gkt is the Drosophila TDP1. Although the mutant is generally healthy and fertile, females exhibit reduced lifespan and diminished climbing ability. This phenotype was rescued by neuronal expression of TDP1. In addition, when c03958 larvae were exposed to bleomycin, an agent that produces oxidative DNA damage, or topoisomerase I-targeted drugs (camptothecin and a noncamptothecin indenoisoquinoline derivative, LMP-776), survivors displayed rough eye patches, which were rescued by neuronal expression of TDP1. Our study establishes that gkt is the Drosophila TDP1 gene, and that it is critical for neuroprotection, normal longevity, and repair of damaged DNA.aging | neuroscience | DNA repair | topoisomerase | fly

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Topoisomerase I and Genome Stability: The Good and the Bad

Cho

Jinks-Robertson

2017

Methods in Molecular Biology

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Topoisomerase I (Top1) resolves torsional stress that accumulates during transcription, replication and chromatin remodeling by introducing a transient single-strand break in DNA. The cleavage activity of Top1 has opposing roles, either promoting or destabilizing genome integrity depending on the context. Resolution of transcription-associated negative supercoils, for example, prevents pairing of the nascent RNA with the DNA template (R-loops) as well as DNA secondary structure formation. Reduced Top1 levels thus enhance CAG repeat contraction, somatic hypermutation, and class switch recombination. Actively transcribed ribosomal DNA is also destabilized in the absence of Top1, reflecting the importance of Top1 in ensuring efficient transcription. In terms of promoting genome instability, an aborted Top1 catalytic cycle stimulates deletions at short tandem repeats and the enzyme's transesterification activity supports illegitimate recombination. Finally, Top1 incision at ribonucleotides embedded in DNA generates deletions in tandem repeats, and induces gross chromosomal rearrangements and mitotic recombination.

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Reversible Top1 cleavage complexes are stabilized strand-specifically at the ribosomal replication fork barrier and contribute to ribosomal DNA stability

Cited by 23 publications

References 54 publications

SUMO Pathway Modulation of Regulatory Protein Binding at the Ribosomal DNA Locus inSaccharomyces cerevisiae

SUMO Pathway Modulation of Regulatory Protein Binding at the Ribosomal DNA Locus inSaccharomyces cerevisiae

Neuroprotection and repair of 3′-blocking DNA ends by glaikit (gkt) encoding Drosophila tyrosyl-DNA phosphodiesterase 1 (TDP1)

Topoisomerase I and Genome Stability: The Good and the Bad

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