Yasuhiro Tsutsui

The Schizosaccharomyces pombe rad60 Gene Is Essential for Repairing Double-Strand DNA Breaks Spontaneously Occurring during Replication and Induced by DNA-Damaging Agents

ABSTRACT To identify novel genes involved in DNA double-strand break (DSB) repair, we previously isolated Schizosaccharomyces pombe mutants which are hypersensitive to methyl methanesulfonate (MMS) and synthetic lethals with rad2. This study characterizes one of these mutants, rad60-1. The gene that complements the MMS sensitivity of this mutant was cloned and designated rad60. rad60 encodes a protein with 406 amino acids which has the conserved ubiquitin-2 motif found in ubiquitin family proteins. rad60-1 is hypersensitive to UV and γ rays, epistatic to rhp51, and defective in the repair of DSBs caused by γ-irradiation. The rad60-1 mutant is also temperature sensitive for growth. At the restrictive temperature (37°C), rad60-1 cells grow for several divisions and then arrest with 2C DNA content; the arrested cells accumulate DSBs and have a diffuse and often aberrantly shaped nuclear chromosomal domain. The rad60-1 mutant is a synthetic lethal with rad18-X, and expression of wild-type rad60 from a multicopy plasmid partially suppresses the MMS sensitivity of rad18-X cells. rad18 encodes a conserved protein of the structural maintenance of chromosomes (SMC) family (A. R. Lehmann, M. Walicka, D. J. Griffiths, J. M. Murray, F. Z. Watts, S. McCready, and A. M. Carr, Mol. Cell. Biol. 15:7067-7080, 1995). These results suggest that S. pombe Rad60 is required to repair DSBs, which accumulate during replication, by recombination between sister chromatids. Rad60 may perform this function in concert with the SMC protein Rad18.

3′ fragment of miR173-programmed RISC-cleaved RNA is protected from degradation in a complex with RISC and SGS3

trans-acting small interfering RNAs (tasiRNAs) are plant-specific endogenous siRNAs produced via a unique pathway whose first step is the microRNA (miRNA)-programmed RNA-induced silencing complex (RISC)–mediated cleavage of tasiRNA gene (TAS) transcripts. One of the products is subsequently transformed into tasiRNAs by a pathway that requires several factors including SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6. Here, using in vitro assembled ARGONAUTE (AGO)1–RISCs, we show that SGS3 is recruited onto RISCs only when they bind target RNA. Following cleavage by miRNA173 (miR173)-programmed RISC, SGS3 was found in complexes containing cleaved TAS2 RNA and RISC. The 3′ cleavage fragment (the source of tasiRNAs) was protected from degradation in this complex. Depletion of SGS3 did not affect TAS2 RNA cleavage by miR173-programmed RISC, but did affect the stability of the 3′ cleavage fragment. When the 3′ nucleotide of 22-nt miR173 was deleted or the corresponding nucleotide in TAS2 RNA was mutated, the complex was not observed and the 3′ cleavage fragment was degraded. Importantly, these changes in miR173 or TAS2 RNA are known to lead to a loss of tasiRNA production in vivo. These results suggest that (i) SGS3 associates with AGO1–RISC via the double-stranded RNA formed by the 3′-terminal nucleotides of 22-nt miR173 and corresponding target RNA, which probably protrudes from the AGO1–RISC molecular surface, (ii) SGS3 protects the 3′ cleavage fragment of TAS2 RNA from degradation, and (iii) the observed SGS3-dependent stabilization of the 3′ fragment of TAS2 RNA is key to tasiRNA production.

Molecular Characterization of the Role of the Schizosaccharomyces pombe nip1/ctp1 Gene in DNA Double-Strand Break Repair in Association with the Mre11-Rad50-Nbs1 Complex

ABSTRACT The Schizosaccharomyces pombe nip1+/ctp1+ gene was previously identified as an slr (synthetically lethal with rad2) mutant. Epistasis analysis indicated that Nip1/Ctp1 functions in Rhp51-dependent recombinational repair, together with the Rad32 (spMre11)-Rad50-Nbs1 complex, which plays important roles in the early steps of DNA double-strand break repair. Nip1/Ctp1 was phosphorylated in asynchronous, exponentially growing cells and further phosphorylated in response to bleomycin treatment. Overproduction of Nip1/Ctp1 suppressed the DNA repair defect of an nbs1-s10 mutant, which carries a mutation in the FHA phosphopeptide-binding domain of Nbs1, but not of an nbs1 null mutant. Meiotic DNA double-strand breaks accumulated in the nip1/ctp1 mutant. The DNA repair phenotypes and epistasis relationships of nip1/ctp1 are very similar to those of the Saccharomyces cerevisiae sae2/com1 mutant, suggesting that Nip1/Ctp1 is a functional homologue of Sae2/Com1, although the sequence similarity between the proteins is limited to the C-terminal region containing the RHR motif. We found that the RxxL and CxxC motifs are conserved in Schizosaccharomyces species and in vertebrate CtIP, originally identified as a cofactor of the transcriptional corepressor CtBP. However, these two motifs are not found in other fungi, including Saccharomyces and Aspergillus species. We propose that Nip1/Ctp1 is a functional counterpart of Sae2/Com1 and CtIP.

Mechanistic insights into the activation of Rad51-mediated strand exchange from the structure of a recombination activator, the Swi5-Sfr1 complex

Rad51 forms a helical filament on single-stranded DNA and promotes strand exchange between two homologous DNA molecules during homologous recombination. The Swi5-Sfr1 complex interacts directly with Rad51 and stimulates strand exchange. Here we describe structural and functional aspects of the complex. Swi5 and the C-terminal core domain of Sfr1 form an essential activator complex with a parallel coiled-coil heterodimer joined firmly together via two previously uncharacterized leucine-zipper motifs and a bundle. The resultant coiled coil is sharply kinked, generating an elongated crescent-shaped structure suitable for transient binding within the helical groove of the Rad51 filament. The N-terminal region of Sfr1, meanwhile, has an interface for binding of Rad51. Our data suggest that the snug fit resulting from the complementary geometry of the heterodimer activates the Rad51 filament and that the N-terminal domain of Sfr1 plays a role in the efficient recruitment of the Swi5-Sfr1 complex to the Rad51 filaments.

Fission Yeast Swi5-Sfr1 Protein Complex, an Activator of Rad51 Recombinase, Forms an Extremely Elongated Dogleg-shaped Structure

Background: The Swi5-Sfr1 protein complex is an activator of Rad51 recombinase, which mediates DNA strand exchange in homologous recombination. Results: Swi5 and Sfr1 form a 1:1 complex, which exhibits an extremely elongated dogleg-shaped structure in solution. Conclusion: The Swi5-Sfr1 structure is suitable for binding within the helical groove of the Rad51 filament. Significance: A structural model will advance our understanding of the molecular mechanism of homologous recombination. In eukaryotes, DNA strand exchange is the central reaction of homologous recombination, which is promoted by Rad51 recombinases forming a right-handed nucleoprotein filament on single-stranded DNA, also known as a presynaptic filament. Accessory proteins known as recombination mediators are required for the formation of the active presynaptic filament. One such mediator in the fission yeast Schizosaccharomyces pombe is the Swi5-Sfr1 complex, which has been identified as an activator of Rad51 that assists in presynaptic filament formation and stimulates its strand exchange reaction. Here, we determined the 1:1 binding stoichiometry between the two subunits of the Swi5-Sfr1 complex using analytical ultracentrifugation and electrospray ionization mass spectrometry. Small-angle x-ray scattering experiments revealed that the Swi5-Sfr1 complex displays an extremely elongated dogleg-shaped structure in solution, which is consistent with its exceptionally high frictional ratio (f/f0) of 2.0 ± 0.2 obtained by analytical ultracentrifugation. Furthermore, we determined a rough topology of the complex by comparing the small-angle x-ray scattering-based structures of the Swi5-Sfr1 complex and four Swi5-Sfr1-Fab complexes, in which the Fab fragments of monoclonal antibodies were specifically bound to experimentally determined sites of Sfr1. We propose a model for how the Swi5-Sfr1 complex binds to the Rad51 filament, in which the Swi5-Sfr1 complex fits into the groove of the Rad51 filament, leading to an active and stable presynaptic filament.

Multiple regulation of Rad51-mediated homologous recombination by fission yeast Fbh1.

Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCFFbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated.

Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.

RNA interference regulates the cell cycle checkpoint through the RNA export factor, Ptr1, in fission yeast

Ago1, an effector protein of RNA interference (RNAi), regulates heterochromatin silencing and cell cycle arrest in fission yeast. However, the mechanism by which Ago1 controls cell cycle checkpoint following hydroxyurea (HU) treatment has not been elucidated. In this study, we show that Ago1 and other RNAi factors control cell cycle checkpoint following HU treatment via a mechanism independent of silencing. While silencing requires dcr1(+), the overexpression of ago1(+) alleviated the cell cycle defect in dcr1Δ. Ago1 interacted with the mRNA export factor, Ptr1. The ptr1-1 mutation impaired cell cycle checkpoint but gene silencing was unaffected. Genetic analysis revealed that the regulation of cell cycle checkpoint by ago1(+) is dependent on ptr1(+). Nuclear accumulation of poly(A)(+) RNAs was detected in mutants of ago1(+) and ptr1(+), suggesting there is a functional link between the cell cycle checkpoint and RNAi-mediated RNA quality control.

In vitro site-specific recombination mediated by the tyrosine recombinase XerA of Thermoplasma acidophilum

In organisms with circular chromosomes, such as bacteria and archaea, an odd number of homologous recombination events can generate a chromosome dimer. Such chromosome dimers cannot be segregated unless they are converted to monomers before cell division. In Escherichia coli, dimer‐to‐monomer conversion is mediated by the paralogous XerC and XerD recombinases at a specific dif site in the replication termination region. Dimer resolution requires the highly conserved cell division protein/chromosome translocase FtsK, and this site‐specific chromosome resolution system is present or predicted in most bacteria. However, most archaea have only XerA, a homologue of the bacterial XerC/D proteins, but no homologues of FtsK. In addition, the molecular mechanism of XerA‐mediated chromosome resolution in archaea has been less thoroughly elucidated than those of the corresponding bacterial systems. In this study, we identified two XerA‐binding sites (dif1 and dif2) in the Thermoplasma acidophilum chromosome. In vitro site‐specific recombination assays showed that dif2, but not dif1, serves as a target site for XerA‐mediated chromosome resolution. Mutational analysis indicated that not only the core consensus sequence of dif2, but also its flanking regions play important roles in the recognition and recombination reactions mediated by XerA.

Human CtIP and Its Homologs: Team Players in DSB Resection Games

Double-strand breaks (DSBs) are among the most serious forms of DNA damage and thus must be efficiently repaired. In order for effective repair to take place, cells must employ the following steps: 1) recognition of the DSBs, 2) cell-cycle arrest via checkpoint activation, and 3) repair of the breaks. In the repair step, DSB ends are nucleolytically processed, which leads to the subsequent recruitment of appropriate repair proteins. Several proteins, including the Mre11 nuclease, are known to be involved in the processing of DSB ends. Additionally, recent studies have identified human CtIP and its orthologs as novel components required for DNA end processing among eukaryotes. This protein is involved not only in repair via homologous recombination (HR) but also in several important biological processes, such as transcriptional regulation and checkpoint control. Importantly, CtIP acts as a tumor suppressor in mammals. In this chapter, we will summarize the existing knowledge on this multi-functional molecule.