Lim15/Dmc1 enhances DNA topoisomerase II catenation activity independent of sequence homology

We reported previously that Coprinus cinereus Lim15/Dmc1 (CcLim15), a meiosis-specific recA-like protein, could specifically activate C. cinereus DNA topoisomerase II (CcTopII). In particular, it enhanced the catenation activity of CcTopII in vitro at the meiotic prophase stage (Iwabata K, Koshiyama A, Yamaguchi T, Sugawara H, Hamada NF, Namekawa HS, Ishii S, Ishizaki T, Chiku H, Nara T, Sakaguchi K, Nucleic Acids Res, 33:5809–5818, 2005). In this study, the interaction between CcTopII and CcLim15, especially during catenation, was investigated in detail using atomic force microscopy. We demonstrated earlier that CcLim15 enhanced the catenation activity of CcTopII in a dose-dependent manner. When using two different-sized plasmid rings (5.4 and 3 kbp), which did not have any homologous sequence regions, equal proportions of homologous and heterologous catenanes were produced, suggesting that CcLim15 causes an increase in catenation activity irrespective of the presence of homologous sequences between the rings. We also showed that CcLim15 works as a DNA-condensing agent. Therefore, we speculate that CcLim15 may work as a DNA-condensing factor specific to the zygotene event and that CcTopII is likely to resolve tangles when the chromosomes initiate pairing at multiple sites by CcLim15.     

Meiosis and small ubiquitin-related modifier (SUMO)-conjugating enzyme, Ubc9

In this review, we describe the role of a small ubiquitin-like protein modifier (SUMO)-conjugating protein, Ubc9, in synaptonemal complex formation during meiosis in a basidiomycete, Coprinus cinereus. Because its meiotic cell cycle is long and naturally synchronous, it is suitable for molecular biological, biochemical and genetic studies of meiotic prophase events. In yeast two-hybrid screening using the meiotic-specific cDNA library of C. cinereus, we found that the meiotic RecA homolog CcLim15 interacted with CcUbc9, CcTopII and CcPCNA. Moreover, both TopII and PCNA homologs were known as Ubc9 interactors and the targets of sumoylation. Immunocytochemistry demonstrates that CcUbc9, CcTopII and CcPCNA localize with CcLim15 in meiotic nuclei during leptotene to zygotene when synaptonemal complex is formed and when homologous chromosomes pair. We discuss the relationships between Lim15/Dmc1 (CcLim15), TopII (CcTopII), PCNA (CcPCNA) and CcUbc9, and subsequently, the role of sumoylation in the stages. We speculate that CcLim15 and CcTopII work in cohesion between homologous chromatins initially and then, in the process of the zygotene events, CcUbc9 works with factors including CcLim15 and CcTopII as an inhibitor of ubiquitin-mediated degradation and as a metabolic switch in the meiotic prophase cell cycle. After CcLim15-CcTopII dissociation, CcLim15 remains on the zygotene DNA and recruits CcUbc9, Rad54B, CcUbc9, Swi5-Sfr1, CcUbc9 and then CcPCNA in rotation on the C-terminus. Finally during zygotene, CcPCNA replaces CcLim15 on the DNA and the free-CcLim15 is probably ubiquitinated and disappears. CcPCNA may recruit the polymerase. The idea that CcUbc9 intervenes in every step by protecting CcLim15 and by switching several factors at the C-terminus of CcLim15 is likely. At the boundary of the zygotene and pachytene stages, CcPCNA would be sumoylated. CcUbc9 may also be involved with CcPCNA in the switch from the replicative polymerase being recruited at zygotene to the repair-type DNA polymerases being recruited at pachytene.     

Sumoylation of a meiosis-specific RecA homolog, Lim15/Dmc1, via interaction with the small ubiquitin-related modifier (SUMO)-conjugating enzyme Ubc9

Sumoylation is a post-translational modification system that covalently attaches the small ubiquitin-related modifier (SUMO) to target proteins. Ubc9 is required as the E2-type enzyme for SUMO-1 conjugation to targets. Here, we show that Ubc9 interacts with the meiosis-specific RecA homolog, Lim15/Dmc1 in the basidiomycete Coprinus cinereus (CcLim15), and mediates sumoylation of CcLim15 during meiosis. In vitro protein-protein interaction assays revealed that CcUbc9 interacts with CcLim15 and binds to the C-terminus (amino acids 105-347) of CcLim15, which includes the ATPase domain. Immunocytochemistry demonstrates that CcUbc9 and CcLim15 colocalize in the nuclei from the leptotene stage to the early pachytene stage during meiotic prophase I. Coimmunoprecipitation experiments indicate that CcUbc9 interacts with CcLim15 in vivo during meiotic prophase I. Furthermore, we show that CcLim15 is a target protein of sumoylation both in vivo and in vitro, and identify the C-terminus (amino acids 105-347) of CcLim15 as the site of sumoylation in vitro. These results suggest that sumoylation is a candidate modulator of meiotic recombination via interaction between Ubc9 and Lim15/Dmc1.     

DNA polymerase mu interacts with a meiosis-specific RecA homolog Lim15 during meiosis in Coprinus cinereus

Meiosis is a fundamental process in eukaryotes. Homologous chromosomes are paired and recombined during meiotic prophase I, which results in variation among the gametes. However, the mechanism of recombination between the maternal and paternal chromosome is unknown. In this study, we report on the identification of interaction between Coprinus cinereus DNA polymerase mu (CcPol mu) and CcLim15/Dmc1, a meiosis-specific RecA-like protein, during meiosis. Interaction between these two proteins was confirmed using a GST-pull down assay. A two-hybrid assay revealed that the N-terminus of CcPol mu, which includes the BRCT domain, is responsible for binding the C-terminus of CcLim15. Furthermore, co-immunoprecipitation experiments indicate that these two proteins also interact in the crude extract of the meiotic cell. A significant proportion of CcPol mu and CcLim15 is shown to co-localize in nuclei from the leptotene/zygotene stage to the early pachytene stage during meiotic prophase I. Moreover, CcLim15 enhances polymerase activity of CcPol mu early in the reaction. These results suggest that CcPol mu might be recruited by CcLim15 and elongate the D-loop structure during homologous recombination in meiosis.     

Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF-1: a molecular link between recombination and chromatin assembly during meiosis

In eukaryotes, meiosis leads to genetically variable gametes through recombination between homologous chromosomes of maternal and paternal origin. Chromatin organization following meiotic recombination is critical to ensure the correct segregation of homologous chromosomes into gametes. However, the mechanism of chromatin organization after meiotic recombination is unknown. In this study we report that the meiosis-specific recombinase Lim15/Dmc1 interacts with the homologue of the largest subunit of chromatin assembly factor 1 (CAF-1) in the basidiomycete Coprinopsis cinerea (Coprinus cinereus). Using C. cinerea LIM15/DMC1 (CcLIM15) as the bait in a yeast two-hybrid screen, we have isolated the C. cinerea homologue of Cac1, the largest subunit of CAF-1 in Saccharomyces cerevisiae, and named it C. cinerea Cac1-like (CcCac1L). Two-hybrid assays confirmed that CcCac1L binds CcLim15 in vivo. beta-Galactosidase assays revealed that the N-terminus of CcCac1L preferentially interacts with CcLim15. Co-immunoprecipitation experiments showed that these proteins also interact in the crude extract of meiotic cells. Furthermore, we demonstrate that, during meiosis, CcCac1L interacts with proliferating cell nuclear antigen (PCNA), a component of the DNA synthesis machinery recently reported as an interacting partner of Lim15/Dmc1. Taken together, these results suggest a novel role of the CAF-1-PCNA complex in meiotic events. We propose that the CAF-1-PCNA complex modulates chromatin assembly following meiotic recombination.     

Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity

PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg(2+) ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.     

Hairpin structures formed by alpha satellite DNA of human centromeres are cleaved by human topoisomerase IIalpha

Although centromere function has been conserved through evolution, apparently no interspecies consensus DNA sequence exists. Instead, centromere DNA may be interconnected through the formation of certain DNA structures creating topological binding sites for centromeric proteins. DNA topoisomerase II is a protein, which is located at centromeres, and enzymatic topoisomerase II activity correlates with centromere activity in human cells. It is therefore possible that topoisomerase II recognizes and interacts with the alpha satellite DNA of human centromeres through an interaction with potential DNA structures formed solely at active centromeres. In the present study, human topoisomerase IIα-mediated cleavage at centromeric DNA sequences was examined in vitro. The investigation has revealed that the enzyme recognizes and cleaves a specific hairpin structure formed by alpha satellite DNA. The topoisomerase introduces a single-stranded break at the hairpin loop in a reaction, where DNA ligation is partly uncoupled from the cleavage reaction. A mutational analysis has revealed, which features of the hairpin are required for topoisomerease IIα-mediated cleavage. Based on this a model is discussed, where topoisomerase II interacts with two hairpins as a mediator of centromere cohesion.     

Fbxo28 promotes mitotic progression and regulates topoisomerase IIα-dependent DNA decatenation

Topoisomerase IIα is an essential enzyme that resolves topological constraints in genomic DNA. It functions in disentangling intertwined chromosomes during anaphase leading to chromosome segregation thus preserving genomic stability. Here we describe a previously unrecognized mechanism regulating topoisomerase IIα activity that is dependent on the F-box protein Fbxo28. We find that Fbxo28, an evolutionarily conserved protein, is required for proper mitotic progression. Interfering with Fbxo28 function leads to a delay in metaphase-to-anaphase progression resulting in mitotic defects as lagging chromosomes, multipolar spindles and multinucleation. Furthermore, we find that Fbxo28 interacts and colocalizes with topoisomerase IIα throughout the cell cycle. Depletion of Fbxo28 results in an increase in topoisomerase IIα?dependent DNA decatenation activity. Interestingly, blocking the interaction between Fbxo28 and topoisomerase IIα also results in multinucleated cells. Our findings suggest that Fbxo28 regulates topoisomerase IIα decatenation activity and plays an important role in maintaining genomic stability.     

Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

Meiosis depends on homologous recombination (HR) in most sexually reproducing organisms. Efficient meiotic HR requires the activity of the meiosis-specific recombinase, Dmc1. Previous work shows Dmc1 is expressed in Entamoeba histolytica, a eukaryotic parasite responsible for amoebiasis throughout the world, suggesting this organism undergoes meiosis. Here, we demonstrate Dmc1 protein is expressed in E. histolytica. We show that purified ehDmc1 forms presynaptic filaments and catalyzes ATP-dependent homologous DNA pairing and DNA strand exchange over at least several thousand base pairs. The DNA pairing and strand exchange activities are enhanced by the presence of calcium and the meiosis-specific recombination accessory factor, Hop2-Mnd1. In combination, calcium and Hop2-Mnd1 dramatically increase the rate of DNA strand exchange activity of ehDmc1. The biochemical system described herein provides a basis on which to better understand the role of ehDmc1 and other HR proteins in E. histolytica.     

Condensin minimizes topoisomerase II‐mediated entanglements of DNA in vivo

The juxtaposition of intracellular DNA segments, together with the DNA‐passage activity of topoisomerase II, leads to the formation of DNA knots and interlinks, which jeopardize chromatin structure and gene expression. Recent studies in budding yeast have shown that some mechanism minimizes the knotting probability of intracellular DNA. Here, we tested whether this is achieved via the intrinsic capacity of topoisomerase II for simplifying the equilibrium topology of DNA; or whether it is mediated by SMC (structural maintenance of chromosomes) protein complexes like condensin or cohesin, whose capacity to extrude DNA loops could enforce dissolution of DNA knots by topoisomerase II. We show that the low knotting probability of DNA does not depend on the simplification capacity of topoisomerase II nor on the activities of cohesin or Smc5/6 complexes. However, inactivation of condensin increases the occurrence of DNA knots throughout the cell cycle. These results suggest an in vivo role for the DNA loop extrusion activity of condensin and may explain why condensin disruption produces a variety of alterations in interphase chromatin, in addition to persistent sister chromatid interlinks in mitotic chromatin.     

Excess single-stranded DNA inhibits meiotic double-strand break repair

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1.We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Δ cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Δ cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.     

DNA Supercoiling Factor Localizes to Puffs on Polytene Chromosomes in Drosophila melanogaster

DNA supercoiling factor (SCF) was first identified in silkworm as a protein that generates negative supercoils in DNA in conjunction with eukaryotic topoisomerase II. To analyze the in vivo role of the factor, we cloned a cDNA encoding Drosophila melanogaster SCF. Northern analysis revealed 1.6- and 1.8-kb mRNAs throughout development. The longer mRNA contains an open reading frame that shares homology with mouse reticulocalbin whereas the shorter one encodes a truncated version lacking the N-terminal signal peptide-like sequence. An antibody against SCF detected a 45-kDa protein in the cytoplasmic fraction and a 30-kDa protein in the nuclear fraction of embryonic extracts. Immunoprecipitation suggests that the 30-kDa protein interacts with topoisomerase II in the nucleus, and hence that it is a functional form of SCF. Immunostaining of blastoderm embryos showed that SCF is present in nuclei during interphase but is excluded from mitotic chromosomes. In larvae, the antibody stained the nuclei of several tissues including a posterior part of the salivary gland. This latter staining was associated with natural or ecdysteroid-induced puffs on polytene chromosomes. Upon heat treatment of larvae, the staining on the endogenous puffs disappeared, and strong staining appeared on heat shock puffs. These results implicate SCF in gene expression.     

An endogenous protein inhibitor,YjhX (TopAI), for topoisomerase I from Escherichia coli

Almost all free-living bacteria contain toxin-antitoxin (TA) systems on their genomes and the targets of toxins are highly diverse. Here, we found a novel, previously unidentified TA system in Escherichia coli named yjhX-yjhQ. Induction of YjhX (85 amino acid residues) causes cell-growth arrest resulting in cell death, while YjhQ (181 residues) co-induction resumes cell growth. The primary cellular target of YjhX was found to be topoisomerase I (TopA), inhibiting both DNA replication and RNA synthesis. Notably, YjhX has no homology to any other toxins of the TA systems. YjhX was expressed well with an N-terminal protein S (PrS) tag in soluble forms. PrS-YjhX specifically interacts with the N-terminal region of TopA (TopA67) but not full-TopA in the absence of plasmid DNA, while PrS-YjhX binds to full-TopA in the presence of DNA. Notably, YjhX does not directly interact with DNA and RNA. YjhX inhibits only topoisomerase I but not topoisomerase III and IV in vitro. Hence, yjhX is renamed as the gene for the TopA inhibitor (the topAI gene). TopAI is the first endogenous protein inhibitor specific for topoisomerase I.     

Purification of GidA protein,a novel topoisomerase II inhibitor produced by Streptomyces flavoviridis

The presence of topoisomerase II inhibition activities in the intracellular extract of Streptomyces flavoviridis was investigated. One active compound inhibiting relaxation activity of topoisomerase II was determined to be a protein. This active principle was purified to homogeneity by gel filtration followed by ion exchange chromatography. The apparent molecular mass was 42 kDa as determined by SDS-PAGE. MALDI TOF peptide mass fingerprinting analysis confirmed this topoisomerase II inhibitor, as glucose-inhibited division protein A (GidA) by MOWSE score of 72. The effects of purified GidA protein on DNA relaxation and decatenation by topoisomerase II were investigated. It inhibited topoisomerase II activity and acted as a topoisomerase poison that significantly stabilized the covalent DNA-topoisomerase II reaction intermediate “cleavable complex”, as observed with etoposide. Collectively, these findings indicate that GidA is a potent inhibitor of topoisomerase II enzyme, which can be exploited for rational drug design in human carcinomas.     

Functional interactions of DNA topoisomerases with a human replication origin

The human DNA replication origin, located in the lamin B2 gene, interacts with the DNA topoisomerases I and II in a cell cycle-modulated manner. The topoisomerases interact in vivo and in vitro with precise bonds ahead of the start sites of bidirectional replication, within the pre-replicative complex region; topoisomerase I is bound in M, early G1 and G1/S border and topoisomerase II in M and the middle of G1. The Orc2 protein competes for the same sites of the origin bound by either topoisomerase in different moments of the cell cycle; furthermore, it interacts on the DNA with topoisomerase II during the assembly of the pre-replicative complex and with DNA-bound topoisomerase I at the G1/S border. Inhibition of topoisomerase I activity abolishes origin firing. Thus, the two topoisomerases are closely associated with the replicative complexes, and DNA topology plays an essential functional role in origin activation.     

Chromosomal integration of LTR-flanked DNA in yeast expressing HIV-1 integrase: down regulation by RAD51

HIV-1 integrase (IN) is the key enzyme catalyzing the proviral DNA integration step. Although the enzyme catalyzes the integration step accurately in vitro, whether IN is sufficient for in vivo integration and how it interacts with the cellular machinery remains unclear. We set up a yeast cellular integration system where integrase was expressed as the sole HIV-1 protein and targeted the chromosomes. In this simple eukaryotic model, integrase is necessary and sufficient for the insertion of a DNA containing viral LTRs into the genome, thereby allowing the study of the isolated integration step independently of other viral mechanisms. Furthermore, the yeast system was used to identify cellular mechanisms involved in the integration step and allowed us to show the role of homologous recombination systems. We demonstrated physical interactions between HIV-1 IN and RAD51 protein and showed that HIV-1 integrase activity could be inhibited both in the cell and in vitro by RAD51 protein. Our data allowed the identification of RAD51 as a novel in vitro IN cofactor able to down regulate the activity of this retroviral enzyme, thereby acting as a potential cellular restriction factor to HIV infection.     

Human Rad54B is a double-stranded DNA-dependent ATPase and has biochemical properties different from its structural homolog in yeast, Tid1/Rdh54

The RAD52 epistasis group genes are involved in homologous recombination, and they are conserved from yeast to humans. We have cloned a novel human gene, RAD54B, which is homologous to yeast and human RAD54. Human Rad54B (hRad54B) shares high homology with human Rad54 (hRad54) in the central region containing the helicase motifs characteristic of the SNF2/SWI2 family of proteins, but the N-terminal domain is less conserved. In yeast, another RAD54 homolog, TID1/RDH54, plays a role in recombination. Tid1/Rdh54 interacts with yeast Rad51 and a meiosis-specific Rad51 homolog, Dmc1. The N-terminal domain of hRad54B shares homology with that of Tid1/Rdh54, suggesting that Rad54B may be the human counterpart of Tid1/Rdh54. We purified the hRad54 and hRad54B proteins from baculovirus-infected insect cells and examined their biochemical properties. hRad54B, like hRad54, is a DNA-binding protein and hydrolyzes ATP in the presence of double-stranded DNA, though its rate of ATP hydrolysis is lower than that of hRad54. Human Rad51 interacts with hRad54 and enhances its ATPase activity. In contrast, neither human Rad51 nor Dmc1 directly interacts with hRad54B. Although hRad54B is the putative counterpart of Tid1/Rdh54, our findings suggest that hRad54B behaves differently from Tid1/Rdh54.     

Topoisomerase activity of the hyperthermophilic replication initiator protein Rep75

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi replicates via the rolling circle mechanism. pGT5 encodes the replication initiator protein Rep75 that exhibits a nicking–closing (NC) activity in vitro on single-stranded oligonucleotides containing the pGT5 double-stranded origin (dso) sequence. Some mesophilic Rep proteins present site-specific DNA topoisomerase-like activity on a negatively supercoiled plasmid harbouring the dso. We report here that Rep75 also exhibits topoisomerase activity on a negatively supercoiled DNA substrate. This DNA topoisomerase-like activity is dependent on the amino acids involved in NC activity of Rep75. However, in contrast with mesophilic Rep proteins, Rep75 topoisomerase activity is not dso dependent. Moreover, although pGT5 is known to be relaxed in vivo, Rep75 was not able to act on a relaxed plasmid in vitro, whether or not it contained the dso.     

Topoisomerase II is a structural component of mitotic chromosome scaffolds

We have obtained a polyclonal antibody that recognizes a major polypeptide component of chicken mitotic chromosome scaffolds. This polypeptide migrates in SDS PAGE with Mr 170,000. Indirect immunofluorescence and subcellular fractionation experiments confirm that it is present in both mitotic chromosomes and interphase nuclei. Two lines of evidence suggest that this protein is DNA topoisomerase II, an abundant nuclear enzyme that controls DNA topological states: anti-scaffold antibody inhibits the strand-passing activity of DNA topoisomerase II; and both anti-scaffold antibody and an independent antibody raised against purified bovine topoisomerase II recognize identical partial proteolysis fragments of the 170,000-mol-wt scaffold protein in immunoblots. Our results suggest that topoisomerase II may be an enzyme that is also a structural protein of interphase nuclei and mitotic chromosomes.