Skb1

[Gabelo Camphire]

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Gross chromosomal rearrangements (GCRs), such as translocations, can occur using repetitive sequences that are abundant and widespread in eukaryotic genomes1. In humans, the total number of repetitive sequences, including satellite repeats and transposable elements, accounts for 54% of the genome2,3. GCRs cause cell death and genetic disorders, including cancer. On the other hand, GCRs can be a driving force of evolution by creating genome diversity4. Therefore, GCRs are not only pathological but also physiological phenomena.

Homologous recombination is required to repair detrimental DNA damage such as double-strand breaks23. Rad51 is the key player in canonical homologous recombination and catalyses homology search and DNA strand exchange, forming displacement loops. Mammalian BRCA1 and BRCA2 facilitate Rad51-dependent recombination, and their mutations increase GCRs and predispose the carriers to cancer24,25. Homologous recombination maintains centromere integrity. In mammals, the inactivation of Rad51 increases aberrant recombination at centromeres9,10,26. In fission yeast, loss of Rad51 increases isochromosome formation at centromeres20,21,27. Detailed analysis using fission yeast showed that Rad51 preferentially promotes a conservative way of recombination: non-crossover recombination at centromeres27,28, thereby suppressing isochromosome formation.

Another recombinase Rad52 promotes homology-dependent DNA recombination/repair independent of Rad5129,30. Rad52, on its own, promotes displacement loop formation, single-strand annealing (SSA), and inverse-strand exchange using RNA strands. Yeast Rad52 also facilitates Rad51 loading onto replication protein A (RPA)-coated single-stranded DNA, while human Rad52 does not have the loader activity31. In both mammals and fission yeast, Rad52-dependent non-canonical recombination causes GCRs at centromeres9,32. In fission yeast, Rad52 causes isochromosome formation via crossover recombination with Mus81, a crossover-specific endonuclease27,32,33,34,35. PCNA ubiquitination at lysine 107 and Msh2-Msh3 have been implicated in the Rad52-dependent GCR pathway32,36. The DNA sliding clamp PCNA may form DNA structures leading to Rad52-dependent GCRs because PCNA K107 is dispensable for DNA damage repair36. The rad52 deletion does not eliminate isochromosome formation, suggesting the presence of a Rad52-independent GCR pathway(s). Moreover, the initial event that leads to GCRs remains unclear.

To gain insights into the GCR mechanism, we search for the factors that cause GCRs in the rad51Δ mutant strain and find Srr1 and Skb1. In A. thaliana and mice, the Srr1 homolog affects the transcription of the genes involved in the circadian rhythm37,38,39. Skb1 is involved in a range of pathways, including cell morphology and cell cycle regulation in fission yeast40,41,42,43, and is the homolog of the human protein arginine methyltransferase 5 (PRMT5)44,45. Srr1 and Skb1 specifically promote isochromosome formation. Remarkably, the srr1 mutation increases DNA damage sensitivity and chromosome loss but is not essential for checkpoint response to DNA damage, suggesting that Srr1 promotes DNA damage repair. srr1 and rad52 mutations additively reduced GCR rates, suggesting that Srr1 and Rad52 have overlapping and non-overlapping roles in GCRs. In contrast to srr1, the skb1 deletion does not increase DNA damage sensitivity and, intriguingly, reduces chromosome loss in rad51Δ cells. Loss of Slf140,41 or Pom142,43, which functions with Skb1 in cell morphology and cell cycle regulation, did not reduce GCRs. However, mutating conserved residues in the arginine methyltransferase (RMTase) domain of Skb1 strongly reduced GCRs, suggesting that Skb1 causes isochromosome formation through its RMTase activity. These findings pave new avenues to decipher the mechanism of GCR events at the centromere.

As Srr1 and Skb1 promote isochromosome formation mediated by centromere repeats, they might be involved in the recombinational repair of DNA damage. To test this possibility, we performed a serial dilution assay and determined the sensitivity of srr1 and skb1 mutant strains to DNA-damaging agents (Fig. 3a). Methyl methanesulfonate (MMS) is a DNA alkylating agent; hydroxyurea (HU) depletes dNTP pool; camptothecin (CPT) is a topoisomerase inhibitor. These agents interfere with the progression of replication forks and create DNA breaks. Compared to wild-type, srr1Δ cells exhibited hypersensitivity to all the DNA-damaging agents (Fig. 3a, top panels). Notably, srr1Δ rad51Δ cells were more sensitive than the single mutants, suggesting a role for Srr1 in Rad51-independent DNA damage response. The srr1-W157R mutation that partially reduced GCR rates (Fig. 1e) also partially increased the damage sensitivity. These results suggest that Srr1 facilitates Rad51-independent DNA damage response.

Skb1 has been implicated in a wide range of pathways, including cell morphology and cell cycle regulation. Skb1 interacts with Slf1 and localizes to cell cortical nodes depending on Slf1, promoting rod-like cell morphology of fission yeast40,41. The DYRK-family kinase Pom1 negatively regulates cell cycle progression to ensure that cells grow to a certain size before entering mitosis42. Genetic evidence shows that Skb1 acts in the Pom1 pathway to regulate the cell cycle independently of its methyltransferase activity43. To ask whether Skb1 promotes isochromosome formation through these pathways, we disrupted slf1 or pom1 genes and determined the GCR rates of the mutant strains (Fig. 6a). Unlike skb1Δ, slf1Δ and pom1Δ slightly increased GCR rates in the wild-type background and neither reduced GCR rates in the rad51Δ background, showing that Skb1 promotes isochromosome formation through the function independent of Slf1 or Pom1.

P.M., N.T., and T.N. conceived the study. P.M., N.T., and Z.P. performed most experiments with technical help from R.X., Y.K., K.O., and T.N. Deep sequencing was performed by H.T., Y.O., and T.H. The manuscript was written by T.N. and P.M. and approved by all the authors.

Communications Biology thanks Gerben Vader, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Manuel Breuer. A peer review file is available.

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The p21-activated kinase, Shk1, is required for cell viability, establishment and maintenance of cell polarity, and proper mating response in the fission yeast, Schizosaccharomyces pombe. Previous genetic studies suggested that a presumptive protein methyltransferase, Skb1, functions as a positive modulator of Shk1. However, unlike Shk1, Skb1 is not required for viability or mating of S. pombe cells and contributes only modestly to the regulation of cell morphology under normal growth conditions. Here we demonstrate that Skb1 plays a more significant role in regulating cell growth and polarity under conditions of hyperosmotic stress. We provide evidence that the inability of skb1Delta cells to properly maintain cell polarity in hyperosmotic conditions results from inefficient subcellular targeting of F-actin. We show that Skb1 localizes to cell ends, sites of septation, and nuclei of S. pombe cells. Hyperosmotic shock results in substantial delocalization of Skb1 from cell ends and nuclei, as well as stimulation of Skb1 protein methyltransferase activity. Taken together, our results demonstrate a new role for Skb1 as a mediator of hyperosmotic stress response in fission yeast. We show that the protein methyltransferase activity of the human Skb1 homolog, Skb1Hs, is also stimulated by hyperosmotic stress in fission yeast, providing evidence for evolutionary conservation of a role for Skb1-related proteins as mediators of hyperosmotic stress response, as well as mechanisms involved in regulating this novel class of protein methyltransferases.

Proper flowering time is important for the growth and development of plants, and both too early and too late flowering impose strong negative influences on plant adaptation and seed yield. Thus, it is vitally important to study the mechanism underlying flowering time control in plants. In a previous study by the authors, genome-wide association analysis was used to screen the candidate gene SISTER OF FCA (SSF) that regulates FLOWERING LOCUS C (FLC), a central gene encoding a flowering suppressor in Arabidopsis thaliana.

In plants, flowering refers to the transition from vegetative to reproductive growth, representing an important turning point in the life cycle of plants. Appropriate flowering is crucial for plant adaptation and reproductive success. When to initiate flowering is determined by a combination of endogenous factors [1] and a multitude of external environmental cues [2,3,4]. Six different main pathways that control flowering have been identified: photoperiod, vernalization, autonomous, gibberellin, ambient temperature, and aging [5,6,7]. These pathways not only respond independently to flowering regulation signals, but also interact with other pathways to form a network that regulates flowering in a coordinated manner. FLOWERING LOCUS C (FLC), FLOWERING LOCUS T (FT), and SUPPRESSOR OF OVEREXPRESSION (SOC1) are regulated. They either activate or suppress the expression of downstream genes regulating inflorescence meristem and flower organs such as LEAFY (LFY), APETALA1 (AP1), and FRUITFULL (FUL) to ensure that flowering is induced at the most appropriate time [8].

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