ABSTRACT
Formation of programmed DNA double-strand breaks is essential for initiating meiotic recombination. Genetic studies on Arabidopsis thaliana and Mus musculus have revealed that assembly of a type IIB topoisomerase VI (Topo VI)-like complex, composed of SPO11 and MTOPVIB, is a prerequisite for generating DNA breaks. However, it remains enigmatic if MTOPVIB resembles its Topo VI subunit B (VIB) ortholog in possessing robust ATPase activity, ability to undergo ATP-dependent dimerization, and activation of SPO11-mediated DNA cleavage. Here, we successfully prepared highly pure A. thaliana MTOPVIB and MTOPVIB-SPO11 complex. Contrary to expectations, our findings highlight that MTOPVIB differs from orthologous Topo VIB by lacking ATP-binding activity and independently forming dimers without ATP. Most significantly, our study reveals that while MTOPVIB lacks the capability to stimulate SPO11-mediated DNA cleavage, it functions as a bona fide DNA-binding protein and plays a substantial role in facilitating the dsDNA binding capacity of the MOTOVIB-SPO11 complex. Thus, we illustrate mechanistic divergence between the MTOPVIB-SPO11 complex and classical type IIB topoisomerases.
Subject(s)
Arabidopsis Proteins , Arabidopsis , DNA Topoisomerases, Type II , Adenosine Triphosphate/metabolism , Arabidopsis/genetics , Arabidopsis/enzymology , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Archaeal Proteins , DNA Breaks, Double-Stranded , DNA Topoisomerases/metabolism , DNA Topoisomerases/genetics , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Endodeoxyribonucleases/metabolism , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/chemistry , Evolution, Molecular , Meiosis , Protein MultimerizationABSTRACT
Meiotic recombination plays dual roles in the evolution and stable inheritance of genomes: Recombination promotes genetic diversity by reassorting variants, and it establishes temporary connections between pairs of homologous chromosomes that ensure their future segregation. Meiotic recombination is initiated by generation of double-strand DNA breaks (DSBs) by the conserved topoisomerase-like protein Spo11. Despite strong conservation of Spo11 across eukaryotic kingdoms, auxiliary complexes that interact with Spo11 complexes to promote DSB formation are poorly conserved. Here, we identify DSB-3 as a DSB-promoting protein in the nematode Caenorhabditis elegans Mutants lacking DSB-3 are proficient for homolog pairing and synapsis but fail to form crossovers. Lack of crossovers in dsb-3 mutants reflects a requirement for DSB-3 in meiotic DSB formation. DSB-3 concentrates in meiotic nuclei with timing similar to DSB-1 and DSB-2 (predicted homologs of yeast/mammalian Rec114/REC114), and DSB-1, DSB-2, and DSB-3 are interdependent for this localization. Bioinformatics analysis and interactions among the DSB proteins support the identity of DSB-3 as a homolog of MEI4 in conserved DSB-promoting complexes. This identification is reinforced by colocalization of pairwise combinations of DSB-1, DSB-2, and DSB-3 foci in structured illumination microscopy images of spread nuclei. However, unlike yeast Rec114, DSB-1 can interact directly with SPO-11, and in contrast to mouse REC114 and MEI4, DSB-1, DSB-2, and DSB-3 are not concentrated predominantly at meiotic chromosome axes. We speculate that variations in the meiotic program that have coevolved with distinct reproductive strategies in diverse organisms may contribute to and/or enable diversification of essential components of the meiotic machinery.
Subject(s)
Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/physiology , DNA Breaks, Double-Stranded , Meiosis/physiology , Animals , Caenorhabditis elegans Proteins/genetics , Computational Biology , Genetic Engineering , Genome , Oocytes/radiation effectsABSTRACT
Most eukaryotes possess two RecA-like recombinases (ubiquitous Rad51 and meiosis-specific Dmc1) to promote interhomolog recombination during meiosis. However, some eukaryotes have lost Dmc1. Given that mammalian and yeast Saccharomyces cerevisiae (Sc) Dmc1 have been shown to stabilize recombination intermediates containing mismatches better than Rad51, we used the Pezizomycotina filamentous fungus Trichoderma reesei to address if and how Rad51-only eukaryotes conduct interhomolog recombination in zygotes with high sequence heterogeneity. We applied multidisciplinary approaches (next- and third-generation sequencing technology, genetics, cytology, bioinformatics, biochemistry, and single-molecule biophysics) to show that T. reesei Rad51 (TrRad51) is indispensable for interhomolog recombination during meiosis and, like ScDmc1, TrRad51 possesses better mismatch tolerance than ScRad51 during homologous recombination. Our results also indicate that the ancestral TrRad51 evolved to acquire ScDmc1-like properties by creating multiple structural variations, including via amino acid residues in the L1 and L2 DNA-binding loops.
Subject(s)
Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Genome, Fungal , Homologous Recombination , Hypocreales/metabolism , Meiosis , Rad51 Recombinase/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Cell Cycle Proteins/genetics , DNA, Single-Stranded , DNA-Binding Proteins/genetics , Fungal Proteins/genetics , Hypocreales/genetics , Rad51 Recombinase/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Dmc1 recombinases are essential to homologous recombination in meiosis. Here, we studied the kinetics of the nucleoprotein filament assembly of Saccharomyces cerevisiae Dmc1 using single-molecule tethered particle motion experiments and in vitro biochemical assay. ScDmc1 nucleoprotein filaments are less stable than the ScRad51 ones because of the kinetically much reduced nucleation step. The lower nucleation rate of ScDmc1 results from its lower single-stranded DNA (ssDNA) affinity, compared to that of ScRad51. Surprisingly, ScDmc1 nucleates mostly on the DNA structure containing the single-stranded and duplex DNA junction with the allowed extension in the 5'-to-3' polarity, while ScRad51 nucleation depends strongly on ssDNA lengths. This nucleation preference is also conserved for mammalian RAD51 and DMC1. In addition, ScDmc1 nucleation can be stimulated by short ScRad51 patches, but not by EcRecA ones. Pull-down experiments also confirm the physical interactions of ScDmc1 with ScRad51 in solution, but not with EcRecA. Our results are consistent with a model that Dmc1 nucleation can be facilitated by a structural component (such as DNA junction and protein-protein interaction) and DNA polarity. They provide direct evidence of how Rad51 is required for meiotic recombination and highlight a regulation strategy in Dmc1 nucleoprotein filament assembly.
Subject(s)
Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Meiosis , Rad51 Recombinase/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Cell Cycle Proteins/genetics , Cytoskeleton/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/genetics , Nucleoproteins/metabolism , Rad51 Recombinase/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Single Molecule Imaging/methodsABSTRACT
Microcephalin 1 (MCPH1) was identified from genetic mutations in patients with primary autosomal recessive microcephaly. In response to DNA double-strand breaks (DSBs), MCPH1 forms damage-induced foci and recruits BRCA2-RAD51 complex, a key component of the DSB repair machinery for homologous recombination (HR), to damage sites. Accordingly, the efficiency of HR is significantly attenuated upon depletion of MCPH1. The biochemical characteristics of MCPH1 and its functional interaction with the HR machinery had remained unclear due to lack of highly purified MCPH1 recombinant protein for functional study. Here, we established a mammalian expression system to express and purify MCPH1 protein. We show that MCPH1 is a bona fide DNA-binding protein and provide direct biochemical analysis of this MCPH family protein. Furthermore, we reveal that MCPH1 directly interacts with RAD51 at multiple contact points, providing evidence for how MCPH1 physically engages with the HR machinery. Importantly, we demonstrate that MCPH1 enhances the stability of RAD51 on single-strand DNA, a prerequisite step for RAD51-mediated recombination. Single-molecule tethered particle motion analysis showed a â¼2-fold increase in the lifetime of RAD51-ssDNA filaments in the presence of MCPH1. Thus, our study demonstrates direct crosstalk between microcephaly protein MCPH1 and the recombination component RAD51 for DSB repair.
Subject(s)
BRCA2 Protein/genetics , Cell Cycle Proteins/genetics , Cytoskeletal Proteins/genetics , Microcephaly/genetics , Rad51 Recombinase/genetics , Cytoskeleton/genetics , DNA Breaks, Double-Stranded , DNA Damage/genetics , DNA Repair/genetics , DNA, Single-Stranded/genetics , DNA-Binding Proteins/genetics , Genomic Instability/genetics , Homologous Recombination/genetics , Humans , Microcephaly/pathology , Nucleoproteins/geneticsABSTRACT
Eukaryotic Rad51 protein is essential for homologous-recombination repair of DNA double-strand breaks. Rad51 recombinases first assemble onto single-stranded DNA to form a nucleoprotein filament, required for function in homology pairing and strand exchange. This filament assembly is the first regulation step in homologous recombination. Rad51 nucleation is kinetically slow, and several accessory factors have been identified to regulate this step. Swi5-Sfr1 (S5S1) stimulates Rad51-mediated homologous recombination by stabilizing Rad51 nucleoprotein filaments, but the mechanism of stabilization is unclear. We used single-molecule tethered particle motion experiments to show that mouse S5S1 (mS5S1) efficiently stimulates mouse RAD51 (mRAD51) nucleus formation and inhibits mRAD51 dissociation from filaments. We also used single-molecule fluorescence resonance energy transfer experiments to show that mS5S1 promotes stable nucleus formation by specifically preventing mRAD51 dissociation. This leads to a reduction of nucleation size from three mRAD51 to two mRAD51 molecules in the presence of mS5S1. Compared with mRAD51, fission yeast Rad51 (SpRad51) exhibits fast nucleation but quickly dissociates from the filament. SpS5S1 specifically reduces SpRad51 disassembly to maintain a stable filament. These results clearly demonstrate the conserved function of S5S1 by primarily stabilizing Rad51 on DNA, allowing both the formation of the stable nucleus and the maintenance of filament length.
Subject(s)
Nuclear Proteins/metabolism , Rad51 Recombinase/metabolism , Animals , DNA , Homologous Recombination/physiology , Mice , Nucleoproteins/metabolism , Schizosaccharomyces/metabolismABSTRACT
Estrogens are among the most concerned emerging contaminants in the wastewater treatment effluent due to their sexual disruption in aquatic wildlife. The use of microalgae for secondary wastewater effluent polishing is a promising approach due to the economic benefit and value-added products. In this study, three microalgae species, including Selenastrum capricornutum, Scenedesmus quadricauda and Chlorella vulgaris were selected to conduct batch experiments to examine important mechanisms, especially the role of algal extracellular organic matter (AEOM) on two selected estrogens (17ß-estradiol, E2 and 17α-ethynylestradiol, EE2) removal. Results showed that estrogens could not be significantly degraded under visible light irradiation and adsorption of estrogens by microalgae was negligible. All three living microalgae cultures have ability to remove E2 and EE2, and Selenastrum capricornutum showed the highest E2 and EE2 removal efficiency of 91% and 83%, respectively, corresponding to the reduction of predicted estrogenic activity of 86%. AEOM from three microalgae cultures could induce photodegradation of estrogens, and AEOM from Selenastrum capricornutum and Chlorella vulgaris achieved 100% of E2 and EE2 removal under visible light irradiation. Fluorescence excitation-emission matrix spectroscopy identified humic/fulvic-like substances in AEOM from three microalgae cultures, which might be responsible for inducing the indirect photolysis of E2 and EE2. Therefore, in the living microalgae cultures, the major estrogens removal mechanisms should include biotransformation as well as AEOM meditated photocatalytic degradation. Since removal rates through photodegradation could be faster than biotransformation, the AEOM mediated photocatalytic degradation can play a potential role to remove emerging contaminants when using microalgae technology for wastewater effluent treatment.
Subject(s)
Chlorella vulgaris/metabolism , Estrogens/metabolism , Water Pollutants, Chemical/metabolism , Biotransformation , Estradiol/metabolism , Estrogens/analysis , Estrone/metabolism , Ethinyl Estradiol/analysis , Ethinyl Estradiol/metabolism , Microalgae/metabolism , Photolysis , Wastewater/chemistry , Water Pollutants, Chemical/analysisABSTRACT
During DNA double-strand break and replication fork repair by homologous recombination, the RAD51 recombinase catalyzes the DNA strand exchange reaction via a helical polymer assembled on single-stranded DNA, termed the presynaptic filament. Our published work has demonstrated a dual function of the SWI5-SFR1 complex in RAD51-mediated DNA strand exchange, namely, by stabilizing the presynaptic filament and maintaining the catalytically active ATP-bound state of the filament via enhancement of ADP release. In this study, we have strived to determine the basis for physical and functional interactions between Mus musculus SWI5-SFR1 and RAD51. We found that SWI5-SFR1 preferentially associates with the oligomeric form of RAD51. Specifically, a C-terminal domain within SWI5 contributes to RAD51 interaction. With specific RAD51 interaction defective mutants of SWI5-SFR1 that we have isolated, we show that the physical interaction is indispensable for the stimulation of the recombinase activity of RAD51. Our results thus help establish the functional relevance of the trimeric RAD51-SWI5-SFR1 complex and provide insights into the mechanistic underpinnings of homology-directed DNA repair in mammalian cells.
Subject(s)
Homologous Recombination/genetics , Nuclear Proteins/chemistry , Rad51 Recombinase/chemistry , Adenosine Triphosphate/metabolism , Animals , DNA Breaks, Double-Stranded , DNA Repair/genetics , DNA Replication/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Mice , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Nuclear Proteins/metabolism , Rad51 Recombinase/metabolismABSTRACT
Sigma (σ) factors are bacterial transcription initiation factors that direct transcription at cognate promoters. The promoters recognized by primary σ are composed of -10 and -35 consensus elements separated by a spacer of 17±1 bp for optimal activity. However, how the optimal promoter spacing is sensed by the primary σ remains unclear. In the present study, we examined this issue using a transcriptionally active Bacillus subtilis N-terminally truncated σA (SND100-σA). The results of the present study demonstrate that SND100-σA binds specifically to both the -10 and -35 elements of the trnS spacing variants, of which the spacer lengths range from 14 to 21 bp, indicating that simultaneous and specific recognition of promoter -10 and -35 elements is insufficient for primary σ to discern the optimal promoter spacing. Moreover, shortening in length of the flexible linker between the two promoter DNA-binding domains of σA also does not enable SND100-σA to sense the optimal promoter spacing. Efficient recognition of optimal promoter spacing by SND100-σA requires core RNAP (RNA polymerase) which reduces the flexibility of simultaneous and specific binding of SND100-σA to both promoter -10 and -35 elements. Thus the discrimination of optimal promoter spacing by σ is core-dependent.
Subject(s)
Bacillus subtilis/genetics , DNA-Directed RNA Polymerases/genetics , Promoter Regions, Genetic , Sigma Factor/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA-Directed RNA Polymerases/metabolism , Regulatory Sequences, Nucleic Acid/genetics , Sigma Factor/chemistry , Sigma Factor/metabolism , Transcription, GeneticABSTRACT
Previous studies have led to a model in which the promoter-specific recognition of prokaryotic transcription initiation factor, sigma (σ), is core dependent. Most σ functions were studied on the basis of this tenet. Here, we provide in vitro evidence demonstrating that the intact Bacillus subtilis primary sigma, σ(A), by itself, is able to interact specifically with promoter deoxyribonucleic acid (DNA), albeit with low sequence selectivity. The core-independent promoter-specific interaction of the σ(A) is -10 specific. However, the promoter -10 specific interaction is unable to allow the σ(A) to discern the optimal promoter spacing. To fulfill this goal, the σ(A) requires assistance from core RNA polymerase (RNAP). The ability of σ, by itself, to interact specifically with promoter might introduce a critical new dimension of study in prokaryotic σ function.
Subject(s)
Bacterial Proteins/metabolism , Promoter Regions, Genetic , Sigma Factor/metabolism , Bacillus subtilis , Bacterial Proteins/isolation & purification , Base Sequence , Binding Sites , DNA/chemistry , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , Protein Binding , Sigma Factor/isolation & purificationABSTRACT
ATP-dependent RAD51 recombinases play an essential role in eukaryotic homologous recombination by catalyzing a four-step process: 1) formation of a RAD51 single-filament assembly on ssDNA in the presence of ATP, 2) complementary DNA strand-exchange, 3) ATP hydrolysis transforming the RAD51 filament into an ADP-bound disassembly-competent state, and 4) RAD51 disassembly to provide access for DNA repairing enzymes. Of these steps, filament dynamics between the ATP- and ADP-bound states, and the RAD51 disassembly mechanism, are poorly understood due to the lack of near-atomic-resolution information of the ADP-bound RAD51-DNA filament structure. We report the cryo-EM structure of ADP-bound RAD51-DNA filaments at 3.1 Å resolution, revealing a unique RAD51 double-filament that wraps around ssDNA. Structural analysis, supported by ATP-chase and time-resolved cryo-EM experiments, reveals a collapsing mechanism involving two four-protomer movements along ssDNA for mechanical transition between RAD51 single- and double-filament without RAD51 dissociation. This mechanism enables elastic change of RAD51 filament length during structural transitions between ATP- and ADP-states.
Subject(s)
Cytoskeleton , DNA, Single-Stranded , Protein Subunits , DNA, Complementary , Homologous Recombination , Adenosine TriphosphateABSTRACT
Replication stress causes replication fork stalling, resulting in an accumulation of single-stranded DNA (ssDNA). Replication protein A (RPA) and CTC1-STN1-TEN1 (CST) complex bind ssDNA and are found at stalled forks, where they regulate RAD51 recruitment and foci formation in vivo. Here, we investigate crosstalk between RPA, CST, and RAD51. We show that CST and RPA localize in close proximity in cells. Although CST stably binds to ssDNA with a high affinity at low ionic strength, the interaction becomes more dynamic and enables facilitated dissociation at high ionic strength. CST can coexist with RPA on the same ssDNA and target RAD51 to RPA-coated ssDNA. Notably, whereas RPA-coated ssDNA inhibits RAD51 activity, RAD51 can assemble a functional filament and exhibit strand-exchange activity on CST-coated ssDNA at high ionic strength. Our findings provide mechanistic insights into how CST targets and tethers RAD51 to RPA-coated ssDNA in response to replication stress.
Subject(s)
Rad51 Recombinase/metabolism , Replication Protein A/metabolism , DNA Replication/genetics , DNA Replication/physiology , Electrophoretic Mobility Shift Assay , HEK293 Cells , HeLa Cells , Humans , Immunoprecipitation , Protein Binding , Rad51 Recombinase/genetics , Replication Protein A/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open "triplet gate" for mismatch tolerance. In support, DMC1-Q244M displayed marked increase in DNA dynamics, leading to unobservable DNA map. MD simulation showed highly dispersive mismatched DNA ensemble in RAD51 but well-converged DNA in DMC1 and RAD51-V273P/D274G. Replacing Loop 1 or Loop 2 residues in DMC1 with RAD51 counterparts enhanced DMC1 fidelity, while reciprocal mutations in RAD51 attenuated its fidelity. Our results show that three Loop 1/Loop 2 residues jointly enact contrasting fidelities of DNA recombinases.
Subject(s)
Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Rad51 Recombinase/chemistry , Rad51 Recombinase/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Cell Cycle Proteins/genetics , Cryoelectron Microscopy , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/genetics , Humans , Mice , Protein Conformation, alpha-Helical , Rad51 Recombinase/genetics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence AlignmentABSTRACT
Polyamines, often elevated in cancer cells, have been shown to promote cell growth and proliferation. Whether polyamines regulate other cell functions remains unclear. Here, we explore whether and how polyamines affect genome integrity. When DNA double-strand break (DSB) is induced in hair follicles by ionizing radiation, reduction of cellular polyamines augments dystrophic changes with delayed regeneration. Mechanistically, polyamines facilitate homologous recombination-mediated DSB repair without affecting repair via non-homologous DNA end-joining and single-strand DNA annealing. Biochemical reconstitution and functional analyses demonstrate that polyamines enhance the DNA strand exchange activity of RAD51 recombinase. The effect of polyamines on RAD51 stems from their ability to enhance the capture of homologous duplex DNA and synaptic complex formation by the RAD51-ssDNA nucleoprotein filament. Our work demonstrates a novel function of polyamines in the maintenance of genome integrity via homology-directed DNA repair.
Subject(s)
Polyamines/metabolism , Rad51 Recombinase/metabolism , Recombinational DNA Repair/physiology , Animals , Cell Line, Tumor , DNA Breaks, Double-Stranded/drug effects , DNA Breaks, Double-Stranded/radiation effects , DNA End-Joining Repair/drug effects , DNA End-Joining Repair/physiology , DNA, Single-Stranded/metabolism , Female , Gamma Rays/adverse effects , HEK293 Cells , Hair Follicle/metabolism , Hair Follicle/radiation effects , Humans , Mice , Mice, Inbred C57BL , Ornithine Decarboxylase/metabolism , Ornithine Decarboxylase/pharmacology , Ornithine Decarboxylase Inhibitors , Phthalazines/pharmacology , Piperazines/pharmacology , Rad51 Recombinase/genetics , Recombinational DNA Repair/drug effectsABSTRACT
Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. In vivo, many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events. Biochemical assays allow detailed investigation of the DNA and protein activities of each step in a repair, recombination or mutagenesis event. Each type of assay is a powerful tool but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.
ABSTRACT
The programmed induction of meiotic DNA double-strand breaks (DSBs) by the evolutionarily conserved SPO-11 protein, which is structurally related to archaeal Topo VIA topoisomerases, triggers meiotic recombination. Identification of several meiosis-specific factors that are required for SPO-11-mediated DSB formation raises the question whether SPO-11 alone can cleave DNA. Here, we have developed procedures to express and purify C. elegans SPO-11 in a soluble, untagged, and monodispersed form. Our biochemical and biophysical analyses demonstrate that SPO-11 is monomeric and binds DNA, double-stranded DNA in particular. Importantly, SPO-11 does not exhibit DNA cleavage activity under a wide range of reaction conditions, suggesting that co-factors are needed for DSB induction activity. Our SPO-11 purification system and the findings reported herein should facilitate future mechanistic studies directed at delineating the mechanism of action of the SPO-11 ensemble in meiotic DSB formation.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , DNA Breaks, Double-Stranded , Endodeoxyribonucleases/genetics , Meiosis/genetics , Amino Acid Sequence , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , DNA/genetics , DNA/metabolism , DNA Repair , Endodeoxyribonucleases/metabolism , Protein Binding , Sequence Homology, Amino AcidABSTRACT
Predicting protein structures and simulating protein folding are two of the most important problems in computational biology today. Simulation methods rely on a scoring function to distinguish the native structure (the most energetically stable) from non-native structures. Decoy databases are collections of non-native structures used to test and verify these functions. We present a method to evaluate and improve the quality of decoy databases by adding novel structures and removing redundant structures. We test our approach on 20 different decoy databases of varying size and type and show significant improvement across a variety of metrics. We also test our improved databases on two popular modern scoring functions and show that for most cases they contain a greater or equal number of native-like structures than the original databases, thereby producing a more rigorous database for testing scoring functions.
Subject(s)
Computational Biology/methods , Databases, Protein , Protein Folding , Proteins/chemistry , Algorithms , Computer Simulation , Protein ConformationABSTRACT
Bacillus subtilis σ(D) is an alternative σ factor that possesses a core-independent promoter -10 element binding specificity despite the lack of a distinct footprint on its cognate promoter. We wished to determine whether this property is common to alternative σ factors. To this end, we over-expressed B. subtilis σ(B) in Escherichia coli and analyzed its DNA binding ability by electrophoretic mobility shift assay and DNase I footprinting. The major complex formed by σ(B) and its cognate promoter DNA is heparin-sensitive. However, in contrast to the -10 element binding specificity observed for B. subtilis σ(D) , the promoter binding of σ(B) is specific for the -35 element. These and other results clearly demonstrate that alternative σ factors possess different promoter-binding characteristics, and make core-independent contributions to recognition of their cognate promoters.
Subject(s)
Bacillus subtilis/genetics , Promoter Regions, Genetic , Sigma Factor/chemistry , Bacterial Proteins , Base Sequence , DNA, Bacterial/genetics , Electrophoretic Mobility Shift Assay , Genes, Bacterial , Operon , Protein Binding , Transcription, GeneticABSTRACT
OBJECTIVE: The presence of reciprocal and Robertsonian chromosomal rearrangement is often related to recurrent miscarriage. Using preimplantation genetic diagnosis, the abortion rate can be decreased. Cases treated at our center were reviewed. MATERIALS AND METHODS: A retrospective analysis for either Robertsonian or reciprocal translocations was performed on all completed cycles of preimplantation genetic diagnosis at our center since the first reported case in 2004 until the end of 2010. Day 3 embryo biopsies were carried out, and the biopsied cell was checked by fluorescent in situ hybridization using relevant informative probes. Embryos with a normal or balanced translocation karyotype were transferred on Day 4. RESULTS: Thirty-eight preimplantation genetic diagnosis cycles involving 17 couples were completed. A total of 450 (82.6%) of the total oocytes were MII oocytes, and 158 (60.0%) of the two-pronuclei embryos were biopsied. In 41.4% of the fluorescent in situ hybridization analyses, the results were either normal or balanced. Embryos were transferred back after 21 cycles. Three babies were born from Robertsonian translocation carriers and another two from reciprocal translocation carriers. The miscarriage rate was 0%. Among the reciprocal translocation group, the live delivery rate was 8.3% per ovum pick-up cycle and 18.2% per embryo transfer cycle. Among the Robertsonian translocation group, the live delivery rate was 14.3% per ovum pick-up cycle and 20.0% per embryo transfer cycle. CONCLUSION: There is a trend whereby the outcome for Robertsonian translocation group carriers is better than that for reciprocal translocation group carriers. Aneuploidy screening may possibly be added in order to improve the outcome, especially for individuals with an advanced maternal age. The emergence of an array-based technology should help improve this type of analysis.
Subject(s)
Abortion, Habitual/genetics , Genetic Testing/methods , In Situ Hybridization, Fluorescence/methods , Preimplantation Diagnosis/methods , Translocation, Genetic , Abnormal Karyotype , Abortion, Habitual/prevention & control , Blastomeres/cytology , Embryo Culture Techniques/methods , Female , Fertilization in Vitro/methods , Humans , Male , Oocytes/cytology , Pregnancy , Pregnancy Outcome , Retrospective StudiesABSTRACT
At initiation of cell division, FtsZ, a tubulin-like GTPase, assembles into a so-called Z-ring structure at the site of division. The formation of Z ring is negatively regulated by EzrA, which ensures only one ring at the midcell per cell cycle. The mechanism leading to the negative regulation of Z-ring formation by EzrA has been analyzed. Our data reveal that the interaction between EzrA and FtsZ not only reduces the GTP-binding ability of FtsZ but also accelerates the rate of GTP hydrolysis, both of which are unfavorable for the polymerization of FtsZ. Moreover, the acceleration in rate of GTP hydrolysis by EzrA is attributed to stabilization of the transition state for GTP hydrolysis and reduction in the affinity of GDP for FtsZ. Clearly, EzrA is able to modify the GTP hydrolysis cycle of FtsZ. On the basis of these results, a model for how EzrA acts to negatively regulate Z-ring formation is proposed.