One-dimensional diffusion of TrpR along DNA enhances its affinity for the operator by chemical ratchet mechanism.

Several DNA-binding proteins show the affinities for their specific DNA sites that positively depend on the length of DNA harboring the sites, i. e. antenna effect. DNA looping can cause the effect for proteins with two or more DNA binding sites, i. e. the looping mechanism. One-dimensional diffusion also has been suggested to cause the effect for proteins with single DNA sites, the diffusion mechanism, which could violate detailed balance. We addressed which mechanism is possible for E. coli TrpR showing 104-fold antenna effect with a single DNA binding site. When a trpO-harboring DNA fragment was connected to a nonspecific DNA with biotin-avidin connection, the otherwise sevenfold antenna effect disappeared. This result denies the looping mechanism with an unknown second DNA binding site. The 3.5-fold repression by TrpR in vivo disappeared when a tight LexA binding site was introduced at various sites near the trpO, suggesting that the binding of LexA blocks one-dimensional diffusion causing the antenna effect. These results are consistent with the chemical ratchet recently proposed for TrpR-trpO binding to solve the deviation from detailed balance, and evidence that the antenna effect due to one-dimensional diffusion exists in cells.

Dependence of DNA length on binding affinity between TrpR and trpO of DNA.

We scrutinize the length dependency of the binding affinity of bacterial repressor TrpR protein to trpO (specific site) on DNA. A footprinting experiment shows that the longer the DNA length, the larger the affinity of TrpR to the specific site on DNA. This effect termed "antenna effect" might be interpreted as follows: longer DNA provides higher probability for TrpR to access to the specific site aided by one-dimensional diffusion along the nonspecific sites of DNA. We show that, however, the antenna effect cannot be explained while detailed balance holds among three kinetic states, that is, free protein/DNA, nonspecific complexes, and specific complex. We propose a working hypothesis that slow degree(s) of freedom in the system switch(es) different potentials of mean force causing transitions among the three states. This results in a deviation from detailed balance on the switching timescale. We then derive a simple reaction diffusion/binding model that describes the antenna effect on TrpR binding to its target operator. Possible scenarios for such slow degree(s) of freedom in TrpR-DNA complex are addressed.

Dual-color-emitting green fluorescent protein from the sea cactus Cavernularia obesa and its use as a pH indicator for fluorescence microscopy.

We isolated and characterized a green fluorescent protein (GFP) from the sea cactus Cavernularia obesa. This GFP exists as a dimer and has absorption maxima at 388 and 498 nm. Excitation at 388 nm leads to blue fluorescence (456 nm maximum) at pH 5 and below, and green fluorescence (507 nm maximum) at pH 7 and above, and the GFP is remarkably stable at pH 4. Excitation at 498 nm leads to green fluorescence (507 nm maximum) from pH 5 to pH 9. We introduced five amino acid substitutions so that this GFP formed monomers rather than dimers and then used this monomeric form to visualize intracellular pH change during the phagocytosis of living cells by use of fluorescence microscopy. The intracellular pH change is visualized by use of a simple long-pass emission filter with single-wavelength excitation, which is technically easier to use than dual-emission fluorescent proteins that require dual-wavelength excitation.

Structural and functional analyses of the DMC1-M200V polymorphism found in the human population.

The M200V polymorphism of the human DMC1 protein, which is an essential, meiosis-specific DNA recombinase, was found in an infertile patient, raising the question of whether this homozygous human DMC1-M200V polymorphism may cause infertility by affecting the function of the human DMC1 protein. In the present study, we determined the crystal structure of the human DMC1-M200V variant in the octameric-ring form. Biochemical analyses revealed that the human DMC1-M200V variant had reduced stability, and was moderately defective in catalyzing in vitro recombination reactions. The corresponding M194V mutation introduced in the Schizosaccharomyces pombe dmc1 gene caused a significant decrease in the meiotic homologous recombination frequency. Together, these structural, biochemical and genetic results provide extensive evidence that the human DMC1-M200V mutation impairs its function, supporting the previous interpretation that this single-nucleotide polymorphism is a source of human infertility.

Stimulation of Dmc1-mediated DNA strand exchange by the human Rad54B protein.

The process of homologous recombination is indispensable for both meiotic and mitotic cell division, and is one of the major pathways for double-strand break (DSB) repair. The human Rad54B protein, which belongs to the SWI2/SNF2 protein family, plays a role in homologous recombination, and may function with the Dmc1 recombinase, a meiosis-specific Rad51 homolog. In the present study, we found that Rad54B enhanced the DNA strand-exchange activity of Dmc1 by stabilizing the Dmc1-single-stranded DNA (ssDNA) complex. Therefore, Rad54B may stimulate the Dmc1-mediated DNA strand exchange by stabilizing the nucleoprotein filament, which is formed on the ssDNA tails produced at DSB sites during homologous recombination.

Stimulation of DNA strand exchange by the human TBPIP/Hop2-Mnd1 complex.

In Saccharomyces cerevisiae, the Hop2 protein forms a complex with the Mnd1 protein and is required for the alignment of homologous chromosomes during meiosis, probably through extensive homology matching between them. The Rad51 and Dmc1 proteins, the eukaryotic RecA orthologs, promote strand exchange and may function in the extensive matching of homology within paired DNA molecules. In the present study, we purified the human TBPIP/Hop2-Mnd1 complex and found that it significantly stimulates the Dmc1- and Rad51-mediated strand exchange. The human Hop2-Mnd1 complex preferentially binds to a three-stranded DNA branch, which mimics the strand-exchange intermediate. These findings are consistent with genetic data, which showed that the Hop2 and Mnd1 proteins are required for homology matching between homologous chromosomes. Therefore, the human TBPIP/Hop2-Mnd1 complex may ensure proper pairing between homologous chromosomes through its stimulation of strand exchange during meiosis.

Role of the N-terminal domain of the human DMC1 protein in octamer formation and DNA binding.

The DMC1 protein, a eukaryotic homologue of RecA that shares significant amino acid identity with RAD51, exhibits two oligomeric DNA binding forms, an octameric ring and a helical filament. In the crystal structure of the octameric ring form, the DMC1 N-terminal domain (1-81 amino acid residues) was highly flexible, with multiple conformations. On the other hand, the N-terminal domain of Rad51 makes specific interactions with the neighboring ATPase domain in the helical filament structure. To gain insights into the functional role of the N-terminal domain of DMC1, we prepared a deletion mutant, DMC1-(82-340), that lacks the N-terminal 81 amino acid residues from the human DMC1 protein. Analytical ultracentrifugation experiments revealed that, whereas full-length DMC1 forms a octamer, DMC1-(82-340) is a heptamer. Furthermore, DNA binding experiments showed that DMC1-(82-340) was completely defective in both single-stranded and double-stranded DNA binding activities. Therefore, the N-terminal domain of DMC1 is required for the formation of the octamer, which may support the proper DNA binding activity of the DMC1 protein.

Mutational analyses of the human Rad51-Tyr315 residue, a site for phosphorylation in leukaemia cells.

The human Rad51 protein, which plays a central role in homologous recombination, catalyses homologous pairing. The Rad51-Tyr315 residue is known to be constitutively phosphorylated in leukaemia cells and is thought to reside within the subunit-subunit interface of the Rad51 filament. To study the function of the Tyr315 residue, we purified five Rad51 mutants, Y315D, Y315E, Y315R, Y315A and Y315F, in which the Tyr315 residue was replaced by Asp, Glu, Arg, Ala and Phe, respectively. Biochemical studies of these Rad51 mutants revealed that the Y315D and Y315E mutants are defective in homologous pairing due to their impaired ssDNA binding, but their dsDNA binding remained unaffected. The Y315D, Y315E and Y315R mutants are defective in dsDNA unwinding, which depends on Rad51-filament formation, suggesting that these mutants are defective in filament formation on dsDNA. Therefore, the Rad51-Tyr315 residue plays important roles in ssDNA binding and filament formation.

Positive role of the mammalian TBPIP/HOP2 protein in DMC1-mediated homologous pairing.

In meiosis, homologous recombination preferentially occurs between homologous chromosomes rather than between sister chromatids, which is opposite to the bias of mitotic recombinational repair. The TBPIP/HOP2 protein is a factor that ensures the proper pairing of homologous chromosomes during meiosis. In the present study, we found that the purified mouse TBPIP/HOP2 protein stimulated homologous pairing catalyzed by the meiotic DMC1 recombinase in vitro. In contrast, TBPIP/HOP2 did not stimulate homologous pairing by RAD51, which is another homologous pairing protein acting in both meiotic and mitotic recombination. The positive effect of TBPIP/HOP2 in the DMC1-mediated homologous pairing was only observed when TBPIP/HOP2 first binds to double-stranded DNA, not to single-stranded DNA, before the initiation of the homologous pairing reaction. Deletion analyses revealed that the C-terminal basic region of TBPIP/HOP2 is required for efficient DNA binding and is also essential for its homologous pairing stimulation activity. Therefore, these results suggest that TBPIP/HOP2 directly binds to DNA and functions as an activator for DMC1 during the homologous pairing step in meiosis.

Structural basis for octameric ring formation and DNA interaction of the human homologous-pairing protein Dmc1.

The human Dmc1 protein, a RecA/Rad51 homolog, is a meiosis-specific DNA recombinase that catalyzes homologous pairing. RecA and Rad51 form helical filaments, while Dmc1 forms an octameric ring. In the present study, we crystallized the full-length human Dmc1 protein and solved the structure of the Dmc1 octameric ring. The monomeric structure of the Dmc1 protein closely resembled those of the human and archaeal Rad51 proteins. In addition to the polymerization motif that was previously identified in the Rad51 proteins, we found another hydrogen bonding interaction at the polymer interface, which could explain why Dmc1 forms stable octameric rings instead of helical filaments. Mutagenesis studies identified the inner and outer basic patches that are important for homologous pairing. The inner patch binds both single-stranded and double-stranded DNAs, while the outer one binds single-stranded DNA. Based on these results, we propose a model for the interaction of the Dmc1 rings with DNA.

Purification of single-strand DNA binding protein from an Escherichia coli lysate using counter-current chromatography, partition and precipitation.

Single-strand DNA binding protein (SSB) from Escherichia coli lysate was purified by counter-current chromatography (CCC) using the ammonium sulfate precipitation method in a coiled column. About 5 ml of E. coli lysate was separated by CCC using a polymer phase system composed of 16% (w/w) polyethylene glycol (PEG) 1000 and 17% (w/w) ammonium sulfate aqueous polymer two-phase solvent system. The precipitation of proteins in the lysate took place in the CCC column, and the SSB protein was eluted in the fraction 51-56. Many other impurities were either eluted immediately after the solvent front or precipitated in the column. The identities of the proteins in the fractions and in the precipitate were confirmed by SDS-polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining.

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.

Homologous pairing and ring and filament structure formation activities of the human Xrcc2*Rad51D complex.

The Xrcc2 and Rad51D/Rad51L3 proteins, which belong to the Rad51 paralogs, are required for homologous recombinational repair (HRR) in vertebrates. The Xrcc2 and Rad51D/Rad51L3 genes, whose products interact with each other, have essential roles in ensuring normal embryonic development. In the present study, we coexpressed the human Xrcc2 and Rad51D/Rad51L3 proteins (Xrcc2 and Rad51D, respectively) in Escherichia coli, and purified the Xrcc2*Rad51D complex to homogeneity. The Xrcc2 small middle dotRad51D complex catalyzed homologous pairing between single-stranded and double-stranded DNA, similar to the function of the Xrcc3*Rad51C complex, which is another complex of the Rad51 paralogs. An electron microscopic analysis showed that Xrcc2*Rad51D formed a multimeric ring structure in the absence of DNA. In the presence of ssDNA, Xrcc2*Rad51D formed a filamentous structure, which is commonly observed among the human homologous pairing proteins, Rad51, Rad52, and Xrcc3*Rad51C.