The Human RNA Polymerase I Transcription Terminator Complex Acts as a Replication Fork Barrier That Coordinates the Progress of Replication with rRNA Transcription Activity.

In S phase, the replication and transcription of genomic DNA need to accommodate each other, otherwise their machineries collide, with chromosomal instability as a possible consequence. Here, we characterized the human replication fork barrier (RFB) that is present downstream from the 47S pre-rRNA gene (ribosomal DNA [rDNA]). We found that the most proximal transcription terminator, Sal box T1, acts as a polar RFB, while the other, Sal box T4/T5, arrests replication forks bidirectionally. The fork-arresting activity at these sites depends on polymerase I (Pol I) transcription termination factor 1 (TTF-1) and a replisome component, TIMELESS (TIM). We also found that the RFB activity was linked to rDNA copies with hypomethylated CpG and coincided with the time that actively transcribed rRNA genes are replicated. Failed fork arrest at RFB sites led to a slowdown of fork progression moving in the opposite direction to rRNA transcription. Chemical inhibition of transcription counteracted this deceleration of forks, indicating that rRNA transcription impedes replication in the absence of RFB activity. Thus, our results reveal a role of RFB for coordinating the progression of replication and transcription activity in highly transcribed rRNA genes.

Rad51 presynaptic filament stabilization function of the mouse Swi5-Sfr1 heterodimeric complex.

Homologous recombination (HR) represents a major error-free pathway to eliminate pre-carcinogenic chromosomal lesions. The DNA strand invasion reaction in HR is mediated by a helical filament of the Rad51 recombinase assembled on single-stranded DNA that is derived from the nucleolytic processing of the primary lesion. Recent studies have found that the human and mouse Swi5 and Sfr1 proteins form a complex that influences Rad51-mediated HR in cells. Here, we provide biophysical evidence that the mouse Swi5-Sfr1 complex has a 1:1 stoichiometry. Importantly, the Swi5-Sfr1 complex, but neither Swi5 nor Sfr1 alone, physically interacts with Rad51 and stimulates Rad51-mediated homologous DNA pairing. This stimulatory effect stems from the stabilization of the Rad51-ssDNA presynaptic filament. Moreover, we provide evidence that the RSfp (rodent Sfr1 proline rich) motif in Sfr1 serves as a negative regulatory element. These results thus reveal an evolutionarily conserved function in the Swi5-Sfr1 complex and furnish valuable information as to the regulatory role of the RSfp motif that is specific to the mammalian Sfr1 orthologs.

Role for the mammalian Swi5-Sfr1 complex in DNA strand break repair through homologous recombination.

In fission yeast, the Swi5-Sfr1 complex plays an important role in homologous recombination (HR), a pathway crucial for the maintenance of genomic integrity. Here we identify and characterize mammalian Swi5 and Sfr1 homologues. Mouse Swi5 and Sfr1 are nuclear proteins that form a complex in vivo and in vitro. Swi5 interacts in vitro with Rad51, the DNA strand-exchange protein which functions during HR. By generating Swi5(-/-) and Sfr1(-/-) embryonic stem cell lines, we found that both proteins are mutually interdependent for their stability. Importantly, the Swi5-Sfr1 complex plays a role in HR when Rad51 function is perturbed in vivo by expression of a BRC peptide from BRCA2. Swi5(-/-) and Sfr1(-/-) cells are selectively sensitive to agents that cause DNA strand breaks, in particular ionizing radiation, camptothecin, and the Parp inhibitor olaparib. Consistent with a role in HR, sister chromatid exchange induced by Parp inhibition is attenuated in Swi5(-/-) and Sfr1(-/-) cells, and chromosome aberrations are increased. Thus, Swi5-Sfr1 is a newly identified complex required for genomic integrity in mammalian cells with a specific role in the repair of DNA strand breaks.

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.

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.

Fission yeast Swi5 protein, a novel DNA recombination mediator.

The Schizosaccharomyces pombe Swi5 protein forms two distinct protein complexes, Swi5-Sfr1 and Swi5-Swi2, each of which plays an important role in the related but functionally distinct processes of homologous recombination and mating-type switching, respectively. The Swi5-Sfr1 mediator complex has been shown to associate with the two RecA-like recombinases, Rhp51 (spRad51) and Dmc1, and to stimulate in vitro DNA strand exchange reactions mediated by these proteins. Genetic analysis indicates that Swi5-Sfr1 works independently of another mediator complex, Rhp55-Rhp57, during Rhp51-dependent recombinational repair. In addition, mutations affecting the two mediators generate distinct repair spectra of HO endonuclease-induced DNA double strand breaks, suggesting that these recombination mediators differently regulate recombination outcomes in an independent manner.

Fission yeast Swi5/Sfr1 and Rhp55/Rhp57 differentially regulate Rhp51-dependent recombination outcomes.

Several accessory proteins referred to as mediators are required for the full activity of the Rad51 (Rhp51 in fission yeast) recombinase. In this study, we analyzed in vivo functions of the recently discovered Swi5/Sfr1 complex from fission yeast. In normally growing cells, the Swi5-GFP protein localizes to the nucleus, where it forms a diffuse nuclear staining pattern with a few distinct foci. These spontaneous foci do not form in swi2Delta mutants. Upon UV irradiation, Swi5 focus formation is induced in swi2Delta mutants, a response that depends on Sfr1 function, and Sfr1 also forms foci that colocalize with damage-induced Rhp51 foci. The number of UV-induced Rhp51 foci is partially reduced in swi5Delta and rhp57Delta mutants and completely abolished in an swi5Delta rhp57Delta double mutant. An assay for products generated by HO endonuclease-induced DNA double-strand breaks (DSBs) reveals that Rhp51 and Rhp57, but not Swi5/Sfr1, are essential for crossover production. These results suggest that Swi5/Sfr1 functions as an Rhp51 mediator but processes DSBs in a manner different from that of the Rhp55/57 mediator.

The Swi5-Sfr1 complex stimulates Rhp51/Rad51- and Dmc1-mediated DNA strand exchange in vitro.

Nucleoprotein filaments made up of Rad51 or Dmc1 recombinases, the core structures of recombination, engage in ATP-dependent DNA-strand exchange. The ability of recombinases to form filaments is enhanced by recombination factors termed 'mediators'. Here, we show that the Schizosaccharomyces pombe Swi5-Sfr1 complex, a conserved eukaryotic protein complex, at substoichiometric concentrations stimulates strand exchange mediated by Rhp51 (the S. pombe Rad51 homolog) and Dmc1 on long DNA substrates. Reactions mediated by both recombinases are completely dependent on Swi5-Sfr1, replication protein A (RPA) and ATP, although RPA inhibits the reaction when it is incubated with single-stranded DNA (ssDNA) before the recombinase. The Swi5-Sfr1 complex overcomes, at least partly, the inhibitory effect of RPA, representing a novel class of mediator. Notably, the Swi5-Sfr1 complex preferentially stimulates the ssDNA-dependent ATPase activity of Rhp51, and it increases the amounts of Dmc1 bound to ssDNA.

The Neurospora crassa mus-19 gene is identical to the qde-3 gene, which encodes a RecQ homologue and is involved in recombination repair and postreplication repair.

An allele called mus-19 was identified by screening temperature-sensitive and mutagen-sensitive mutants of Neurospora crassa. The mus-19 gene was genetically mapped to a region near the end of the right arm of linkage group I, where a RecQ homologue called qde-3 had been physically mapped in the Neurospora database. Complementation tests between the mus-19 mutant and the qde-3(RIP) mutant showed that mus-19 and qde-3 were the same gene. The qde-3 genes of both mutants were cloned and sequenced; and the results showed that they have mutation(s) in their qde-3 genes. The original mus-19 and qde-3(RIP) mutants are defective in quelling, as reported for other qde-3 mutants. The mutants show high sensitivity to methyl methanesulfonate, ethyl methanesulfonate, N-methyl- N'-nitro- N-nitrosoguanidine, tert-butyl hydroperoxide, 4-nitroquinoline-1-oxide, hydroxyurea and histidine. Epistasis analysis indicated that the qde-3 gene belongs both to the uvs-6 recombination repair pathway and the uvs-2 postreplication repair pathway. The qde-3 mutation has no effect on the integration of a plasmid carrying the mtr gene by homologous recombination. In homozygous crosses, the qde-3 mutant is defective in ascospore production.

Two different Swi5-containing protein complexes are involved in mating-type switching and recombination repair in fission yeast.

Homologous recombination is an important biological process that occurs in all organisms and facilitates genome rearrangements and repair of DNA double-strand breaks. Eukaryotic Rad51 proteins (Rad51sp or Rhp51 in fission yeast) are functional and structural homologs of bacterial RecA protein, an evolutionarily conserved protein that plays a key role in homologous pairing and strand exchange between homologous DNA molecules in vitro. Here we show that the fission yeast swi5+ gene, which was originally identified as a gene required for normal mating-type switching, encodes a protein conserved among eukaryotes and is involved in a previously uncharacterized Rhp51 (Rad51sp)-dependent recombination repair pathway that does not require the Rhp55/57 (Rad55/57sp) function. Protein interactions with both Swi5 and Rhp51 were found to be mediated by a domain common to Swi2 and Sfr1 (Swi five-dependent recombination repair protein 1, a previously uncharacterized protein with sequence similarity to the C-terminal part of Swi2). Genetic epistasis analyses suggest that the Swi5-Sfr1-Rhp51 interactions function specifically in DNA recombination repair, whereas the Swi5-Swi2-Rhp51 interactions may function, together with chromodomain protein Swi6 (HP1 homolog), in mating-type switching.

Molecular characterization of the Schizosaccharomyces pombe nbs1+ gene involved in DNA repair and telomere maintenance.

The human MRN complex is a multisubunit nuclease that is composed of Mre11, Rad50, and Nbs1 and is involved in homologous recombination and DNA damage checkpoints. Mutations of the MRN genes cause genetic disorders such as Nijmegen breakage syndrome. Here we identified a Schizosaccharomyces pombe nbs1(+) homologue by screening for mutants with mutations that caused methyl methanesulfonate (MMS) sensitivity and were synthetically lethal with the rad2Delta mutation. Nbs1 physically interacts with the C-terminal half of Rad32, the Schizosaccharomyces pombe Mre11 homologue, in a yeast two-hybrid assay. nbs1 mutants showed sensitivities to gamma-rays, UV, MMS, and hydroxyurea and displayed telomere shortening similar to the characteristics of rad32 and rad50 mutants. nbs1, rad32, and rad50 mutant cells were elongated and exhibited abnormal nuclear morphology. These findings indicate that S. pombe Nbs1 forms a complex with Rad32-Rad50 and is required for homologous recombination repair, telomere length regulation, and the maintenance of chromatin structure. Amino acid sequence features and some characteristics of the DNA repair function suggest that the S. pombe Rad32-Rad50-Nbs1 complex has functional similarity to the corresponding MRN complexes of higher eukaryotes. Therefore, S. pombe Nbs1 will provide an additional model system for studying the molecular function of the MRN complex associated with genetic diseases.

Competition between the Rad50 complex and the Ku heterodimer reveals a role for Exo1 in processing double-strand breaks but not telomeres.

The Mre11-Rad50-Nbs1(Xrs2) complex and the Ku70-Ku80 heterodimer are thought to compete with each other for binding to DNA ends. To investigate the mechanism underlying this competition, we analyzed both DNA damage sensitivity and telomere overhangs in Schizosaccharomyces pombe rad50-d, rad50-d pku70-d, rad50-d exo1-d, and pku70-d rad50-d exo1-d cells. We found that rad50 exo1 double mutants are more methyl methanesulfonate (MMS) sensitive than the respective single mutants. The MMS sensitivity of rad50-d cells was suppressed by concomitant deletion of pku70+. However, the MMS sensitivity of the rad50 exo1 double mutant was not suppressed by the deletion of pku70+. The G-rich overhang at telomere ends in taz1-d cells disappeared upon deletion of rad50+, but the overhang reappeared following concomitant deletion of pku70+. Our data suggest that the Rad50 complex can process DSB ends and telomere ends in the presence of the Ku heterodimer. However, the Ku heterodimer inhibits processing of DSB ends and telomere ends by alternative nucleases in the absence of the Rad50-Rad32 protein complex. While we have identified Exo1 as the alternative nuclease targeting DNA break sites, the identity of the nuclease acting on the telomere ends remains elusive.