Biology subjects

Freon, K.

Publications and source records attributed to Freon, K..

4 recordsLinked to original sources

bioRxiv2024

The fission yeast SUMO-targeted Ubiquitin Ligase Slx8 functionally associates with clustered centromeres and the silent mating type region at the nuclear periphery

The SUMO-targeted Ubiquitin ligase (STUbL) family is involved in multiple cellular processes via a wide range of mechanisms to maintain genome stability. One of the evolutionarily conserved functions of STUbL is to promote changes in the nuclear positioning of DNA lesions, targeting them to the nuclear periphery. In Schizossacharomyces pombe, the STUbL Slx8 is a regulator of SUMOylated proteins and promotes replication stress tolerance by counteracting the toxicity of SUMO conjugates. In order to study the dynamic dialectic between Ubiquitinylation and SUMOylation in the nuclear space of the S. pombe genome, we analyzed Slx8 localization. Unexpectedly, we did not detect replication stress-induced Slx8 foci. However, we discovered that Slx8 forms a single nuclear focus, enriched at the nuclear periphery, which marks both clustered centromeres at the spindle pole body and the silent mating type region. The formation of this single Slx8 focus requires the E3 SUMO ligase Pli1, poly-SUMOylation and the histone methyl transferase Clr4 that is responsible for the heterochromatin histone mark H3-K9 methylation. Finally, we established that Slx8 promotes centromere clustering and gene silencing at heterochromatin domains. Altogether, our data highlight evolutionarily conserved and functional relationships between STUbL and heterochromatin domains to promote gene silencing and nuclear organization. HighlightsO_LIThe S. pombe STUbL Slx8 forms a single nuclear focus enriched at the nuclear periphery in a SUMO-chain-dependent manner. C_LIO_LISlx8 foci mark clustered centromeres and the silenced mating type region but not telomeres. C_LIO_LIH3-K9 methylation by Crl4 promotes the single nuclear Slx8 focus C_LIO_LISlx8 promotes centromere clustering and gene silencing. C_LI

cell biology↗

bioRxiv2023

SUMO protease and proteasome recruitment at the nuclear periphery differently affect replication dynamics at arrested forks.

Nuclear pores complexes (NPCs) are genome organizers, defining a particular nuclear compartment enriched for SUMO protease and proteasome activities, and acting as docking sites for DNA repair. In fission yeast, the anchorage of perturbed replication forks to NPCs is an integral part of the recombination-dependent replication restart mechanism (RDR) that resumes DNA synthesis at terminally dysfunctional forks. By mapping DNA polymerase usage, we report that SUMO protease Ulp1-associated NPCs ensure efficient initiation of restarted DNA synthesis, whereas proteasome-associated NPCs sustain the progression of restarted DNA polymerase. In contrast to Ulp1-dependent events, this last function occurs independently of SUMO chains formation. By analyzing the role of the nuclear basket, the nucleoplasmic extension of the NPC, we reveal that the activities of Ulp1 and the proteasome cannot compensate for each other and affect RDR dynamics in distinct ways. Our work probes the mechanisms by which the NPC environment ensures optimal RDR. Highlights[bullet] Ulp1-associated NPCs ensure efficient initiation of restarted DNA synthesis, in a SUMO chain-dependent manner [bullet]Proteasome-associated NPCs foster the progression of restarted DNA synthesis, in a SUMO chain-independent manner [bullet]The nucleoporin Nup60 promotes the spatial sequestration of Ulp1 at the nuclear periphery [bullet]Ulp1 and proteasome activities are differently required for optimal recombination-mediated fork restart.

molecular biology↗

bioRxiv2023

Disordered regions and folded modules in CAF-1 promote histone deposition in S. pombe

Genome and epigenome integrity in eukaryotes depends on the proper coupling of histone deposition with DNA synthesis. This process relies on the evolutionary conserved histone chaperone CAF-1 for which the links between structure and functions are still a puzzle. While studies of the S. cerevisiae CAF-1 complex enabled to propose a model for the histone deposition mechanism, we still lack a framework to demonstrate its generality and in particular, how its interaction with the polymerase accessory factor PCNA is operating. Here, we reconstituted a complete SpCAF-1 from fission yeast. We characterized its dynamic structure using NMR, SAXS and molecular modeling together with in vitro and in vivo functional studies on rationally designed interaction mutants. Importantly, we identify the unfolded nature of the acidic domain which folds up when binding to histones. We also show how the long KER helix mediates DNA binding and stimulates SpCAF-1 association with PCNA. Our study highlights how the organization of CAF-1 comprising both disordered regions and folded modules enables the dynamics of multiple interactions to promote synthesis-coupled histone deposition essential for its DNA replication, heterochromatin maintenance, and genome stability functions.

biochemistry↗

bioRxiv2017

The non-homologous end joining factor Ku orchestrates replication fork resection and fine-tunes Rad51-mediated fork restart

Replication requires Homologous Recombination (HR) to stabilize and restart terminally-arrested forks. HR-mediated fork processing requires single stranded DNA (ssDNA) gaps and not necessarily Double Strand Breaks. We used genetic and molecular assays to investigate fork-resection and restart at dysfunctional, unbroken forks in Schizosaccharomyces pombe. We found that fork-resection is a two-step process coordinated by the non-homologous end joining factor Ku. An initial resection mediated by MRN/Ctp1 removes Ku from terminally-arrested forks, generating ~ 110 bp sized gaps obligatory for subsequent Exo1-mediated long-range resection and replication restart. The lack of Ku results in slower fork restart, excessive resection, and impaired RPA recruitment. We propose that terminally-arrested forks undergo fork reversal, providing a single DNA end for Ku binding which primes RPA-coated ssDNA. We uncover an unprecedented role for Ku in orchestrating resection of unbroken forks and in fine-tuning HR-mediated replication restart.\n\nO_LIKu orchestrates a two-steps DNA end-resection of terminally-arrested and unbroken forks\nC_LIO_LIMRN/Ctp1 removes Ku from terminally-arrested forks to initiate fork-resection\nC_LIO_LIa ~110 bp sized ssDNA gap is sufficient and necessary to promote fork restart.\nC_LIO_LIThe lack of Ku decreases ssDNA RPA-coating, and slows down replication fork restart.\nC_LI

genetics↗