Analysis of nitrogen source assimilation in industrial strains of Aspergillus oryzae.

Nitrogen source assimilation is important for the biological functions of fungi, and its pathway has been deeply studied. Aspergillus oryzae mutants defective in nitrogen source assimilation are known to grow poorly on Czapek-Dox (CD) medium. In this study, we found an industrial strain of A. oryzae that grew very poorly on a CD medium containing sodium nitrate as a nitrogen source. We used media with various nitrogen components to examine the steps affecting the nitrogen source assimilation pathway of this strain. The strain grew well on the CD medium supplied with nitrite salt or ammonium salt, suggesting that the strain was defective in nitrate assimilation step. To ascertain the gene causing the defect of nitrate assimilation, a gene expression vector harboring either niaD or crnA of A. oryzae RIB40 was introduced into the industrial strain. The industrial strain containing the crnA vector recovered its growth. This is the first report that a mutation of crnA causes poor growth on CD medium in an industrial strain of A. oryzae, and crnA can be used as a transformation marker for crnA deficient strains.

Fission yeast Srr1 and Skb1 promote isochromosome formation at the centromere.

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.

Preparation of egg-koji for developing a novel food.

While chicken eggs contain many nutrients necessary for humans and there are various cooking methods, the nutritional components are used as they are, and there are no traditional foods that utilize microorganisms. Koji-mold, containing Aspergillus oryzae, A. sojae, and A. luchuensis, which has been used in various fermented foods since ancient times, grows on raw grain materials such as rice and barley to become koji. This can give flavors not found in the raw materials that can decompose and convert the nutritional components of the raw materials. Here, we succeeded for the first time in developing egg-koji that uses only eggs and koji-mold by selecting and combining cooked egg powder (CEP) and A. oryzae AO101 as the most suitable combination. To suppress the explosive growth of harmful bacteria, we improved the sterilization method, watering method, and amount of water. In addition, it was found that egg-koji has a characteristic enzyme activity balance, in which amylase is extremely low and protease at pH 6 was high compared to grain koji, such as rice and barley. Egg-koji might produce enzymes suitable for taking in nutrients when growing into CEP and would be expected to give a flavor that could not be achieved by cooking or additives.

Gross Chromosomal Rearrangement at Centromeres.

Centromeres play essential roles in the faithful segregation of chromosomes. CENP-A, the centromere-specific histone H3 variant, and heterochromatin characterized by di- or tri-methylation of histone H3 9th lysine (H3K9) are the hallmarks of centromere chromatin. Contrary to the epigenetic marks, DNA sequences underlying the centromere region of chromosomes are not well conserved through evolution. However, centromeres consist of repetitive sequences in many eukaryotes, including animals, plants, and a subset of fungi, including fission yeast. Advances in long-read sequencing techniques have uncovered the complete sequence of human centromeres containing more than thousands of alpha satellite repeats and other types of repetitive sequences. Not only tandem but also inverted repeats are present at a centromere. DNA recombination between centromere repeats can result in gross chromosomal rearrangement (GCR), such as translocation and isochromosome formation. CENP-A chromatin and heterochromatin suppress the centromeric GCR. The key player of homologous recombination, Rad51, safeguards centromere integrity through conservative noncrossover recombination between centromere repeats. In contrast to Rad51-dependent recombination, Rad52-mediated single-strand annealing (SSA) and microhomology-mediated end-joining (MMEJ) lead to centromeric GCR. This review summarizes recent findings on the role of centromere and recombination proteins in maintaining centromere integrity and discusses how GCR occurs at centromeres.

Fission yeast Rad8/HLTF facilitates Rad52-dependent chromosomal rearrangements through PCNA lysine 107 ubiquitination.

Gross chromosomal rearrangements (GCRs), including translocation, deletion, and inversion, can cause cell death and genetic diseases such as cancer in multicellular organisms. Rad51, a DNA strand exchange protein, suppresses GCRs by repairing spontaneous DNA damage through a conservative way of homologous recombination, gene conversion. On the other hand, Rad52 that catalyzes single-strand annealing (SSA) causes GCRs using homologous sequences. However, the detailed mechanism of Rad52-dependent GCRs remains unclear. Here, we provide genetic evidence that fission yeast Rad8/HLTF facilitates Rad52-dependent GCRs through the ubiquitination of lysine 107 (K107) of PCNA, a DNA sliding clamp. In rad51Δ cells, loss of Rad8 eliminated 75% of the isochromosomes resulting from centromere inverted repeat recombination, showing that Rad8 is essential for the formation of the majority of isochromosomes in rad51Δ cells. Rad8 HIRAN and RING finger mutations reduced GCRs, suggesting that Rad8 facilitates GCRs through 3' DNA-end binding and ubiquitin ligase activity. Mms2 and Ubc4 but not Ubc13 ubiquitin-conjugating enzymes were required for GCRs. Consistent with this, mutating PCNA K107 rather than the well-studied PCNA K164 reduced GCRs. Rad8-dependent PCNA K107 ubiquitination facilitates Rad52-dependent GCRs, as PCNA K107R, rad8, and rad52 mutations epistatically reduced GCRs. In contrast to GCRs, PCNA K107R did not significantly change gene conversion rates, suggesting a specific role of PCNA K107 ubiquitination in GCRs. PCNA K107R enhanced temperature-sensitive growth defects of DNA ligase I cdc17-K42 mutant, implying that PCNA K107 ubiquitination occurs when Okazaki fragment maturation fails. Remarkably, K107 is located at the interface between PCNA subunits, and an interface mutation D150E bypassed the requirement of PCNA K107 and Rad8 ubiquitin ligase for GCRs. These data suggest that Rad8-dependent PCNA K107 ubiquitination facilitates Rad52-dependent GCRs by changing the PCNA clamp structure.

DNA replication machinery prevents Rad52-dependent single-strand annealing that leads to gross chromosomal rearrangements at centromeres.

Homologous recombination between repetitive sequences can lead to gross chromosomal rearrangements (GCRs). At fission yeast centromeres, Rad51-dependent conservative recombination predominantly occurs between inverted repeats, thereby suppressing formation of isochromosomes whose arms are mirror images. However, it is unclear how GCRs occur in the absence of Rad51 and how GCRs are prevented at centromeres. Here, we show that homology-mediated GCRs occur through Rad52-dependent single-strand annealing (SSA). The rad52-R45K mutation, which impairs SSA activity of Rad52 protein, dramatically reduces isochromosome formation in rad51 deletion cells. A ring-like complex Msh2-Msh3 and a structure-specific endonuclease Mus81 function in the Rad52-dependent GCR pathway. Remarkably, mutations in replication fork components, including DNA polymerase α and Swi1/Tof1/Timeless, change the balance between Rad51-dependent recombination and Rad52-dependent SSA at centromeres, increasing Rad52-dependent SSA that forms isochromosomes. Our results uncover a role of DNA replication machinery in the recombination pathway choice that prevents Rad52-dependent GCRs at centromeres.

Transcriptional silencing of centromere repeats by heterochromatin safeguards chromosome integrity.

The centromere region of chromosomes consists of repetitive DNA sequences, and is, therefore, one of the fragile sites of chromosomes in many eukaryotes. In the core region, the histone H3 variant CENP-A forms centromere-specific nucleosomes that are required for kinetochore formation. In the pericentromeric region, histone H3 is methylated at lysine 9 (H3K9) and heterochromatin is formed. The transcription of pericentromeric repeats by RNA polymerase II is strictly repressed by heterochromatin. However, the role of the transcriptional silencing of the pericentromeric repeats remains largely unclear. Here, we focus on the chromosomal rearrangements that occur at the repetitive centromeres, and highlight our recent studies showing that transcriptional silencing by heterochromatin suppresses gross chromosomal rearrangements (GCRs) at centromeres in fission yeast. Inactivation of the Clr4 methyltransferase, which is essential for the H3K9 methylation, increased GCRs with breakpoints located in centromeric repeats. However, mutations in RNA polymerase II or the transcription factor Tfs1/TFIIS, which promotes restart of RNA polymerase II following its backtracking, reduced the GCRs that occur in the absence of Clr4, demonstrating that heterochromatin suppresses GCRs by repressing the Tfs1-dependent transcription. We also discuss how the transcriptional restart gives rise to chromosomal rearrangements at centromeres.

Heterochromatin suppresses gross chromosomal rearrangements at centromeres by repressing Tfs1/TFIIS-dependent transcription.

Heterochromatin, characterized by histone H3 lysine 9 (H3K9) methylation, assembles on repetitive regions including centromeres. Although centromeric heterochromatin is important for correct segregation of chromosomes, its exact role in maintaining centromere integrity remains elusive. Here, we found in fission yeast that heterochromatin suppresses gross chromosomal rearrangements (GCRs) at centromeres. Mutations in Clr4/Suv39 methyltransferase increased the formation of isochromosomes, whose breakpoints were located in centromere repeats. H3K9A and H3K9R mutations also increased GCRs, suggesting that Clr4 suppresses centromeric GCRs via H3K9 methylation. HP1 homologs Swi6 and Chp2 and the RNAi component Chp1 were the chromodomain proteins essential for full suppression of GCRs. Remarkably, mutations in RNA polymerase II (RNAPII) or Tfs1/TFIIS, the transcription factor that facilitates restart of RNAPII after backtracking, specifically bypassed the requirement of Clr4 for suppressing GCRs. These results demonstrate that heterochromatin suppresses GCRs by repressing Tfs1-dependent transcription of centromere repeats.

Shelterin promotes tethering of late replication origins to telomeres for replication-timing control.

DNA replication initiates at many discrete loci on eukaryotic chromosomes, and individual replication origins are regulated under a spatiotemporal program. However, the underlying mechanisms of this regulation remain largely unknown. In the fission yeast Schizosaccharomyces pombe, the telomere-binding protein Taz1, ortholog of human TRF1/TRF2, regulates a subset of late replication origins by binding to the telomere-like sequence near the origins. Here, we showed using a lacO/LacI-GFP system that Taz1-dependent late origins were predominantly localized at the nuclear periphery throughout interphase, and were localized adjacent to the telomeres in the G1/S phase. The peripheral localization that depended on the nuclear membrane protein Bqt4 was not necessary for telomeric association and replication-timing control of the replication origins. Interestingly, the shelterin components Rap1 and Poz1 were required for replication-timing control and telomeric association of Taz1-dependent late origins, and this requirement was bypassed by a minishelterin Tpz1-Taz1 fusion protein. Our results suggest that Taz1 suppresses replication initiation through shelterin-mediated telomeric association of the origins at the onset of S phase.

Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1.

Post-replicative correction of replication errors by the mismatch repair (MMR) system is critical for suppression of mutations. Although the MMR system may need to handle nucleosomes at the site of chromatin replication, how MMR occurs in the chromatin environment remains unclear. Here, we show that nucleosomes are excluded from a >1-kb region surrounding a mismatched base pair in Xenopus egg extracts. The exclusion was dependent on the Msh2-Msh6 mismatch recognition complex but not the Mlh1-containing MutL homologs and counteracts both the HIRA- and CAF-1 (chromatin assembly factor 1)-mediated chromatin assembly pathways. We further found that the Smarcad1 chromatin remodeling ATPase is recruited to mismatch-carrying DNA in an Msh2-dependent but Mlh1-independent manner to assist nucleosome exclusion and that Smarcad1 facilitates the repair of mismatches when nucleosomes are preassembled on DNA. In budding yeast, deletion of FUN30, the homolog of Smarcad1, showed a synergistic increase of spontaneous mutations in combination with MSH6 or MSH3 deletion but no significant increase with MSH2 deletion. Genetic analyses also suggested that the function of Fun30 in MMR is to counteract CAF-1. Our study uncovers that the eukaryotic MMR system has an ability to exclude local nucleosomes and identifies Smarcad1/Fun30 as an accessory factor for the MMR reaction.

Regulation of mitotic recombination between DNA repeats in centromeres.

Centromeres that are essential for faithful segregation of chromosomes consist of unique DNA repeats in many eukaryotes. Although recombination is under-represented around centromeres during meiosis, little is known about recombination between centromere repeats in mitotic cells. Here, we compared spontaneous recombination that occurs between ade6B/ade6X inverted repeats integrated at centromere 1 (cen1) or at a non-centromeric ura4 locus in fission yeast. Remarkably, distinct mechanisms of homologous recombination (HR) were observed in centromere and non-centromere regions. Rad51-dependent HR that requires Rad51, Rad54 and Rad52 was predominant in the centromere, whereas Rad51-independent HR that requires Rad52 also occurred in the arm region. Crossovers between inverted repeats (i.e. inversions) were under-represented in the centromere as compared to the arm region. While heterochromatin was dispensable, Mhf1/CENP-S, Mhf2/CENP-X histone-fold proteins and Fml1/FANCM helicase were required to suppress crossovers. Furthermore, Mhf1 and Fml1 were found to prevent gross chromosomal rearrangements mediated by centromere repeats. These data for the first time uncovered the regulation of mitotic recombination between DNA repeats in centromeres and its physiological role in maintaining genome integrity.

Rad51 and Rad54 promote noncrossover recombination between centromere repeats on the same chromatid to prevent isochromosome formation.

Centromeres consist of DNA repeats in many eukaryotes. Non-allelic homologous recombination (HR) between them can result in gross chromosomal rearrangements (GCRs). In fission yeast, Rad51 suppresses isochromosome formation that occurs between inverted repeats in the centromere. However, how the HR enzyme prevents homology-mediated GCRs remains unclear. Here, we provide evidence that Rad51 with the aid of the Swi/Snf-type motor protein Rad54 promotes non-crossover recombination between centromere repeats to prevent isochromosome formation. Mutations in Rad51 and Rad54 epistatically increased the rates of isochromosome formation and chromosome loss. In sharp contrast, these mutations decreased gene conversion between inverted repeats in the centromere. Remarkably, analysis of recombinant DNAs revealed that rad51 and rad54 increase the proportion of crossovers. In the absence of Rad51, deletion of the structure-specific endonuclease Mus81 decreased both crossovers and isochromosomes, while the cdc27/pol32-D1 mutation, which impairs break-induced replication, did not. We propose that Rad51 and Rad54 promote non-crossover recombination between centromere repeats on the same chromatid, thereby suppressing crossover between non-allelic repeats on sister chromatids that leads to chromosomal rearrangements. Furthermore, we found that Rad51 and Rad54 are required for gene silencing in centromeres, suggesting that HR also plays a role in the structure and function of centromeres.

MutSα maintains the mismatch repair capability by inhibiting PCNA unloading.

Eukaryotic mismatch repair (MMR) utilizes single-strand breaks as signals to target the strand to be repaired. DNA-bound PCNA is also presumed to direct MMR. The MMR capability must be limited to a post-replicative temporal window during which the signals are available. However, both identity of the signal(s) involved in the retention of this temporal window and the mechanism that maintains the MMR capability after DNA synthesis remain unclear. Using Xenopus egg extracts, we discovered a mechanism that ensures long-term retention of the MMR capability. We show that DNA-bound PCNA induces strand-specific MMR in the absence of strand discontinuities. Strikingly, MutSα inhibited PCNA unloading through its PCNA-interacting motif, thereby extending significantly the temporal window permissive to strand-specific MMR. Our data identify DNA-bound PCNA as the signal that enables strand discrimination after the disappearance of strand discontinuities, and uncover a novel role of MutSα in the retention of the post-replicative MMR capability.

The DNA damage checkpoint pathway promotes extensive resection and nucleotide synthesis to facilitate homologous recombination repair and genome stability in fission yeast.

DNA double-strand breaks (DSBs) can cause chromosomal rearrangements and extensive loss of heterozygosity (LOH), hallmarks of cancer cells. Yet, how such events are normally suppressed is unclear. Here we identify roles for the DNA damage checkpoint pathway in facilitating homologous recombination (HR) repair and suppressing extensive LOH and chromosomal rearrangements in response to a DSB. Accordingly, deletion of Rad3(ATR), Rad26ATRIP, Crb2(53BP1) or Cdc25 overexpression leads to reduced HR and increased break-induced chromosome loss and rearrangements. We find the DNA damage checkpoint pathway facilitates HR, in part, by promoting break-induced Cdt2-dependent nucleotide synthesis. We also identify additional roles for Rad17, the 9-1-1 complex and Chk1 activation in facilitating break-induced extensive resection and chromosome loss, thereby suppressing extensive LOH. Loss of Rad17 or the 9-1-1 complex results in a striking increase in break-induced isochromosome formation and very low levels of chromosome loss, suggesting the 9-1-1 complex acts as a nuclease processivity factor to facilitate extensive resection. Further, our data suggest redundant roles for Rad3ATR and Exo1 in facilitating extensive resection. We propose that the DNA damage checkpoint pathway coordinates resection and nucleotide synthesis, thereby promoting efficient HR repair and genome stability.

Telomere-binding protein Taz1 controls global replication timing through its localization near late replication origins in fission yeast.

In eukaryotes, the replication of chromosome DNA is coordinated by a replication timing program that temporally regulates the firing of individual replication origins. However, the molecular mechanism underlying the program remains elusive. Here, we report that the telomere-binding protein Taz1 plays a crucial role in the control of replication timing in fission yeast. A DNA element located proximal to a late origin in the chromosome arm represses initiation from the origin in early S phase. Systematic deletion and substitution experiments demonstrated that two tandem telomeric repeats are essential for this repression. The telomeric repeats recruit Taz1, a counterpart of human TRF1 and TRF2, to the locus. Genome-wide analysis revealed that Taz1 regulates about half of chromosomal late origins, including those in subtelomeres. The Taz1-mediated mechanism prevents Dbf4-dependent kinase (DDK)-dependent Sld3 loading onto the origins. Our results demonstrate that the replication timing program in fission yeast uses the internal telomeric repeats and binding of Taz1.

DNA polymerization-independent functions of DNA polymerase epsilon in assembly and progression of the replisome in fission yeast.

DNA polymerase epsilon (Pol ε) synthesizes the leading strands, following the CMG (Cdc45, Mcm2-7, and GINS [Go-Ichi-Nii-San]) helicase that translocates on the leading-strand template at eukaryotic replication forks. Although Pol ε is essential for the viability of fission and budding yeasts, the N-terminal polymerase domain of the catalytic subunit, Cdc20/Pol2, is dispensable for viability, leaving the following question: what is the essential role(s) of Pol ε? In this study, we investigated the essential roles of Pol ε using a temperature-sensitive mutant and a recently developed protein-depletion (off-aid) system in fission yeast. In cdc20-ct1 cells carrying mutations in the C-terminal domain of Cdc20, the CMG components, RPA, Pol α, and Pol δ were loaded onto replication origins, but Cdc45 did not translocate from the origins, suggesting that Pol ε is required for CMG helicase progression. In contrast, depletion of Cdc20 abolished the loading of GINS and Cdc45 onto origins, indicating that Pol ε is essential for assembly of the CMG complex. These results demonstrate that Pol ε plays essential roles in both the assembly and progression of CMG helicase.

The prereplication complex recruits XEco2 to chromatin to promote cohesin acetylation in Xenopus egg extracts.

BACKGROUND: Sister chromatids are held together by the ring-shaped cohesin complex, which is loaded onto chromosomes before DNA replication. Cohesion between sister chromosomes is established during DNA replication, and it requires acetylation of the Smc3 subunit of cohesin by evolutionally conserved cohesin acetyltransferases (CoATs). However, how CoATs are recruited to chromatin and how cohesin acetylation is regulated remain unclear. RESULTS: We found that cohesin acetylation requires pre-RC-dependent chromatin loading of cohesin, but surprisingly, it is independent of DNA synthesis in Xenopus egg extracts. Immunodepletion experiments revealed that XEco2 is the CoAT responsible for Smc3 acetylation and sister chromatid cohesion. Recruitment of XEco2 onto chromatin was dependent on pre-RC assembly but was independent of cohesin loading and DNA synthesis. Two short N-terminal motifs, PBM-A and PBM-B, which are conserved among vertebrate Esco2/XEco2 homologs, were collectively essential for pre-RC-dependent chromatin association of XEco2, cohesin acetylation, and subsequent sister chromatid cohesion. The conserved PCNA-interacting protein box in XEco2 was largely dispensable for Smc3 acetylation but was partially required for cohesion. Interaction of acetylated cohesin with DNA was stabilized against salt-wash treatments after DNA replication. CONCLUSIONS: Our results demonstrate that pre-RC formation regulates chromatin association of XEco2 in Xenopus egg extracts. We propose that this reaction is critical to acetylate cohesin, whose DNA binding is subsequently stabilized by DNA replication.

Mcm10 plays an essential role in origin DNA unwinding after loading of the CMG components.

The CMG complex composed of Mcm2-7, Cdc45 and GINS is postulated to be the eukaryotic replicative DNA helicase, whose activation requires sequential recruitment of replication proteins onto Mcm2-7. Current models suggest that Mcm10 is involved in assembly of the CMG complex, and in tethering of DNA polymerase α at replication forks. Here, we report that Mcm10 is required for origin DNA unwinding after association of the CMG components with replication origins in fission yeast. A combination of promoter shut-off and the auxin-inducible protein degradation (off-aid) system efficiently depleted cellular Mcm10 to <0.5% of the wild-type level. Depletion of Mcm10 did not affect origin loading of Mcm2-7, Cdc45 or GINS, but impaired recruitment of RPA and DNA polymerases. Mutations in a conserved zinc finger of Mcm10 abolished RPA loading after recruitment of Mcm10. These results show that Mcm10, together with the CMG components, plays a novel essential role in origin DNA unwinding through its zinc-finger function.

Abundance of prereplicative complexes (Pre-RCs) facilitates recombinational repair under replication stress in fission yeast.

Mcm2-7 complexes are loaded onto chromatin with the aid of Cdt1 and Cdc18/Cdc6 and form prereplicative complexes (pre-RCs) at multiple sites on each chromosome. Pre-RCs are essential for DNA replication and surviving replication stress. However, the mechanism by which pre-RCs contribute to surviving replication stress is largely unknown. Here, we isolated the fission yeast mcm6-S1 mutant that was hypersensitive to methyl methanesulfonate (MMS) and camptothecin (CPT), both of which cause forks to collapse. The mcm6-S1 mutation impaired the interaction with Cdt1 and decreased the binding of minichromosome maintenance (MCM) proteins to replication origins. Overexpression of Cdt1 restored MCM binding and suppressed the sensitivity to MMS and CPT, suggesting that the Cdt1-Mcm6 interaction is important for the assembly of pre-RCs and the repair of collapsed forks. MMS-induced Chk1 phosphorylation and Rad22/Rad52 focus formation occurred normally, whereas cells containing Rhp54/Rad54 foci, which are involved in DNA strand exchange and dissociation of the joint molecules, were increased. Remarkably, G(1) phase extension through deletion of an S phase cyclin, Cig2, as well as Cdt1 overexpression restored pre-RC assembly and suppressed Rhp54 accumulation. A cdc18 mutation also caused hypersensitivity to MMS and CPT and accumulation of Rhp54 foci. These data suggest that an abundance of pre-RCs facilitates a late step in the recombinational repair of collapsed forks in the following S phase.