Biology subjects

Kolesar, P.

Publications and source records attributed to Kolesar, P..

4 recordsLinked to original sources

bioRxiv2025

Homologous recombination mutant lethality differs between h- and h+ Schizosaccharomyces pombe strains due to mat1 heterochromatin

Homologous recombination (HR) is generally considered dispensable in yeast and vertebrates, yet mounting evidence indicates that its essentiality depends on cellular context. Here, we dissect the basis of this context dependency in Schizosaccharomyces pombe. In the homothallic h90 strain, regarded as wild type, mating-type switching (MTS) occurs every other cell division and requires HR to repair programmed double-strand breaks (DSBs) at the mat1 locus. We show that the widely used heterothallic h-S strain is likewise dependent on HR for viability. HR-deficient h-S mutants (rad51{Delta}, rad52{Delta}, or rad54{Delta}), still frequently employed in the literature, survive only when carrying secondary suppressor mutations that abolish mat1 DSB formation, such as smt-0, swi1{Delta}, or fml1{Delta}. In contrast, HR is dispensable in the h+N strain, where duplication of the mat2/3 region into mat1 introduces the cenH and REIII elements. These elements nucleate H3K9 methylation and heterochromatin spreading across the imprint site, blocking imprintosome recruitment and thereby preventing both imprinting and DSB formation. Disruption of this heterochromatin, via deletion of cenH or key chromatin modifiers, restores DSB formation in h+N cells and reinstates HR essentiality in the absence of the Clr4 methyltransferase. Collectively, our findings demonstrate that HR is indispensable for S. pombe survival due to its critical role in repairing mat1 DSBs, except under genetic or epigenetic conditions that suppress their formation.

molecular biology↗

bioRxiv2022

SAGA histone acetyltransferase module facilitates chromatin accessibility to SMC5/6

Structural Maintenance of Chromosomes (SMC) complexes are molecular machines driving chromatin organization at higher levels. In eukaryotes, three SMC complexes (cohesin, condensin, and SMC5/6) play key roles in cohesion, condensation, replication, transcription and DNA repair. Here, we performed a genetic screen in fission yeast to identify novel factors required for SMC5/6 binding to DNA. We identified 79 genes of which histone acetyltransferases (HATs) were the most represented. Genetic and phenotypic analyses suggested a particularly strong functional relationship between the SMC5/6 and SAGA complexes. Furthermore, several SMC5/6 subunits physically interacted with SAGA HAT module components Gcn5 and Ada2. As Gcn5-dependent acetylation facilitates the accessibility of chromatin to DNA repair proteins, we first analysed the formation of DNA damage-induced SMC5/6 foci in the {Delta}gcn5 mutant. The SMC5/6 foci formed normally in {Delta}gcn5, suggesting SAGA-independent SMC5/6 localization to DNA-damaged sites. Next, we used Nse4-FLAG chromatin-immunoprecipitation (ChIP-seq) analysis in unchallenged cells to assess SMC5/6 distribution. A significant portion of SMC5/6 accumulated within gene regions in wild-type cells, which was reduced in {Delta}gcn5 and {Delta}ada2 mutants. The drop in SMC5/6 levels was also observed in gcn5-E191Q acetyltransferase-dead mutant. Altogether, our data suggest that the SAGA HAT module may facilitate chromatin accessibility to SMC5/6 at gene regions. Author SummaryGenomes of all eukaryotes must be folded and packed into their relatively small nuclear spaces. Histones first pack free genomic DNA into nucleosomes and their arrays. Other complexes like histone modifiers and remodelers can regulate nucleosome positions and their packing within chromatin fibres. They assist in the relative opening or condensation of chromatin fibres and facilitate their accessibility to DNA-binding proteins. The highly conserved Structural Maintenance of Chromosomes (SMC) complexes (cohesin, condensin, and SMC5/6) compact further chromatin fibres at higher levels. These molecular machines can loop chromatin fibres, which need access to segments of free DNA for their physical binding to DNA. Here, we studied genetic and physical interactions between histone-modifying SAGA complex and SMC5/6. We show that the SAGA histone acetyltransferase module may facilitate chromatin access to SMC5/6.

genetics↗

bioRxiv2022

Characterization of the NSE6 subunit of the Physcomitrium patens PpSMC5/6 complex

Structural Maintenance of Chromosome (SMC) complexes are molecular machines ensuring chromatin organization at higher levels. They play direct roles in cohesion, condensation, replication, transcription and DNA repair. Their cores are composed of long-armed SMC, kleisin, and kleisin-associated KITE or HAWK subunits. Additional factors, like NSE6 within SMC5/6, bind to SMC core complexes and regulate their activities. To characterize the NSE6 subunit of moss Physcomitrium patens, we analyzed its protein-protein interactions and Ppnse6 mutant phenotypes. We identified a previously unrecognized sequence motif conserved from yeast to humans within the NSE6 CANIN domain that is required for interaction with its NSE5 partner. In addition, the CANIN domain and its preceding sequences bind and link SMC5 and SMC6 arms, suggesting its role in SMC5/6 dynamics. Both Ppnse6dCas9_3 and Ppnse6KO1_47 mutant lines exhibited reduced growth and developmental aberrations. These mutants were also sensitive to DNA-damaging drug bleomycin and lost a significant portion of rDNA copies, suggesting conserved architecture and functions of SMC5/6 complexes across species.

molecular biology↗

bioRxiv2019

A role of the Nse4 kleisin and Nse1/Nse3 KITE subunits in the ATPase cycle of SMC5/6

The SMC (Structural Maintenance of Chromosomes) complexes are composed of SMC dimers, kleisin and kleisin-interacting subunits. Mutual interactions of these subunits constitute the basal architecture of the SMC complexes. Particularly, terminal domains of the kleisin subunit bridge the SMC head domains of the SMC molecules. Binding of ATP molecules to the heads and their hydrolysis alter the shape of long SMC molecules (from rod-like to open-ring shapes) and power them with motor activity. The kleisin-interacting (HAWK or KITE) subunits bind kleisin linker regions and regulate such dynamic activities.\n\nWe developed new systems to follow the interactions between SMC5/6 subunits and stability of the SMC5/6 complexes. First, we show that the N-terminal domain of the Nse4 kleisin molecule binds to the SMC6 neck and bridges it to the SMC5 head. Second, binding of the Nse1 and Nse3 KITE proteins to the Nse4 linker part increases stability of the ATP-free SMC5/6 complex. In contrast, binding of ATP to the SMC5/6 complex containing KITE subunits significantly decreases its stability. Elongation of the Nse4 linker partially suppresses instability of the ATP-bound complex, suggesting that the binding of the KITE proteins to the Nse4 linker constrains its limited size. Our data suggest that the KITE proteins may shape the Nse4 linker to fit the ATP-free complex optimally and to facilitate opening of the complex upon ATP binding. This mechanism suggests an important role of the KITE subunits in the dynamics of the SMC5/6 complexes.\n\nAUTHOR SUMMARYThe SMC5/6 complex is member of the Structural maintenance of chromosomes (SMC) family, key organizers of both prokaryotic and eukaryotic genomes. Their architecture and dynamics (driven by ATP binding and hydrolysis) are essential for cellular processes, like chromatin segregation, condensation, replication and repair. In this paper, we described conserved mode of the Nse4 kleisin subunit binding to the SMC6 (similar to cohesin and condensin) and its bridging role. Furthermore, we showed different impact of the binding of the Nse1-Nse3 KITE subunits to the Nse4 kleisin bridge. Our study suggested that the KITE proteins modulate the stability of the SMC5/6 complex, depending on its binding and hydrolysis of ATP. Our findings uncover molecular mechanisms underlying dynamics of the SMC5/6 complexes.

genetics↗