論文の公開元へFission yeast Srr1 and Skb1 promote isochromosome formation at the centromere
概要
Title
Fission yeast Srr1 and Skb1 promote
isochromosome formation at the centromere
Author(s)
Mongia Piyusha; Toyofuku, Naoko; Pan, Ziyi et
al.
Citation
Communications Biology. 2023, 6, p. 551
Version Type VoR
URL
rights
https://hdl.handle.net/11094/92490
This article is licensed under a Creative
Commons Attribution 4.0 International License.
Note
Osaka University Knowledge Archive : OUKA
https://ir.library.osaka-u.ac.jp/
Osaka University
ARTICLE
https://doi.org/10.1038/s42003-023-04925-9
OPEN
Fission yeast Srr1 and Skb1 promote
isochromosome formation at the centromere
1234567890():,;
Piyusha Mongia 1,2,6, Naoko Toyofuku1,6, Ziyi Pan 1,2,6, Ran Xu1,2, Yakumo Kinoshita1,2, Keitaro Oki1,
Hiroki Takahashi3, Yoshitoshi Ogura4, Tetsuya Hayashi 5 & Takuro Nakagawa 1,2 ✉
Rad51 maintains genome integrity, whereas Rad52 causes non-canonical homologous
recombination leading to gross chromosomal rearrangements (GCRs). Here we find that
fission yeast Srr1/Ber1 and Skb1/PRMT5 promote GCRs at centromeres. Genetic and physical
analyses show that srr1 and skb1 mutations reduce isochromosome formation mediated by
centromere inverted repeats. srr1 increases DNA damage sensitivity in rad51 cells but does
not abolish checkpoint response, suggesting that Srr1 promotes Rad51-independent DNA
repair. srr1 and rad52 additively, while skb1 and rad52 epistatically reduce GCRs. Unlike srr1 or
rad52, skb1 does not increase damage sensitivity. Skb1 regulates cell morphology and cell
cycle with Slf1 and Pom1, respectively, but neither Slf1 nor Pom1 causes GCRs. Mutating
conserved residues in the arginine methyltransferase domain of Skb1 greatly reduces GCRs.
These results suggest that, through arginine methylation, Skb1 forms aberrant DNA structures leading to Rad52-dependent GCRs. This study has uncovered roles for Srr1 and Skb1 in
GCRs at centromeres.
1 Department of Biological Sciences, Graduate School of Science, Osaka University, 1−1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan. 2 Forefront
Research Center, Graduate School of Science, Osaka University, 1−1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan. 3 Medical Mycology Research
Center, Chiba University, Chiba 260-8673, Japan. 4 Division of Microbiology, Department of Infectious Medicine, Kurume University School of Medicine,
Kurume, Fukuoka 830-0011, Japan. 5 Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. 6These authors
contributed equally: Piyusha Mongia, Naoko Toyofuku, Ziyi Pan. ✉email: nakagawa.takuro.sci@osaka-u.ac.jp
COMMUNICATIONS BIOLOGY | (2023)6:551 | https://doi.org/10.1038/s42003-023-04925-9 | www.nature.com/commsbio
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ARTICLE
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COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04925-9
ross 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.
The centromere that ensures proper chromosome segregation
contains repetitive DNA sequences in many eukaryotes. Human
centromeres (≥ 3 Mb) contain α satellite and other types of satellite
repeats, transposable elements, and segmental duplications5. The
orientation of the centromere repeats including higher-order
repeats of α satellites, switches within a centromere, forming
inverted DNA repeats. Despite the important role in chromosome
segregation, the centromere is a hotspot for chromosomal breakage
and rearrangement6–10. Recombination between repetitive
sequences at the centromere forms abnormal chromosomes11–13.
Robertsonian translocations, a fusion of two acrocentric chromosomes at or around centromeres, are the most frequently observed
form of chromosomal abnormality in humans, affecting 1 out of
1000 newborns14. Isochromosomes whose arms are mirror images
of each other are commonly found in cancer cells15. Isochromosomes of chr21 and chrX cause Down and Turner syndromes,
respectively16,17. Compared to mammalian centromeres, the fission
yeast S. pombe centromeres are short (35~110 kb) but contain
inverted DNA repeats flanking a non-repetitive core sequence18,19.
In this fungus, isochromosomes are produced using inverted DNA
repeats in the centromere20–22. Less complexity of the centromere
DNA sequence makes fission yeast an excellent system to study the
mechanism of centromeric GCRs.
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 crossoverspecific endonuclease27,32–35. 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
2
and Skb1. In A. thaliana and mice, the Srr1 homolog affects the
transcription of the genes involved in the circadian rhythm37–39.
Skb1 is involved in a range of pathways, including cell morphology and cell cycle regulation in fission yeast40–43, 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.
Results
Srr1 and Skb1 cause gross chromosomal rearrangements
(GCRs). To gain insights into the mechanism of GCRs, we
introduced random mutations into rad51Δ cells that show elevated GCR rates and searched for the clones that exhibit reduced
levels of GCRs. To detect otherwise lethal GCRs in haploid cells,
we used an extra-chromosome ChLC (~530 kb) derived from
fission yeast chromosome 3 (chr3) and detected spontaneous
GCRs that had lost ura4+ and ade6+ marker genes
(Fig. 1a)20,27,46. To assess GCR rates, yeast clones grown on
Edinburgh minimum media supplemented with uracil and adenine (EMM+UA) were replicated onto the media containing
5-fluoroorotic acid (5-FOA+UA) that is toxic to ura4+ cells. Of
24,000 clones, three reproducibly exhibited reduced levels of
GCRs. Genome sequencing of one of them identified the srr1/
ber1-W157R and skb1-A377V mutations in their SRR1-like and
arginine methyltransferase (RMTase) domains, respectively
(Fig. 1b). The srr1 and skb1 genes are only 51 kb apart from each
other on chr2. The replica plating assay shows that, compared to
the parental rad51Δ strain, the rad51Δ clone containing the srr1
and skb1 mutations from the reduced number of colonies on the
5-FOA+UA plate (Fig. 1c).
To establish whether Srr1 or Skb1 is required for GCRs, we
deleted the genes and determined GCR rates by the fluctuation
test (Fig. 1d). In the wild-type background, srr1Δ but not skb1Δ
slightly reduced GCR rates, showing that Srr1 is required for
GCRs even in the presence of Rad51. To our surprise, not only
srr1Δ but also skb1Δ reduced GCR rates in the rad51Δ
background, demonstrating that both Srr1 and Skb1 cause GCRs.
Remarkably, srr1Δ skb1Δ double mutation further reduced GCRs
than the single mutations, suggesting that Srr1 and Skb1 have
non-overlapping roles in GCRs (see below). ...
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