Katsumi Morimatsu

Inhibition of Escherichia coli RecA coprotease activities by DinI.

In Escherichia coli, the SOS response is induced upon DNA damage and results in the enhanced expression of a set of genes involved in DNA repair and other functions. The initial step, self‐cleavage of the LexA repressor, is promoted by the RecA protein which is activated upon binding to single‐stranded DNA. In this work, induction of the SOS response by the addition of mitomycin C was found to be prevented by overexpression of the dinI gene. dinI is an SOS gene which maps at 24.6 min of the E.coli chromosome and encodes a small protein of 81 amino acids. Immunoblotting analysis with anti‐LexA antibodies revealed that LexA did not undergo cleavage in dinI‐overexpressed cells after UV irradiation. In addition, the RecA‐dependent conversion of UmuD to UmuD′ (the active form for mutagenesis) was also inhibited in dinI‐overexpressed cells. Conversely, a dinI‐deficient mutant showed a slightly faster and more extensive processing of UmuD and hence higher mutability than the wild‐type. Finally, we demonstrated, by using an in vitro reaction with purified proteins, that DinI directly inhibits the ability of RecA to mediate self‐cleavage of UmuD.

Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy

To get mechanistic insight into the DNA strand-exchange reaction of homologous recombination, we solved a filament structure of a human Rad51 protein, combining molecular modeling with experimental data. We build our structure on reported structures for central and N-terminal parts of pure (uncomplexed) Rad51 protein by aid of linear dichroism spectroscopy, providing angular orientations of substituted tyrosine residues of Rad51-dsDNA filaments in solution. The structure, validated by comparison with an electron microscopy density map and results from mutation analysis, is proposed to represent an active solution structure of the nucleo-protein complex. An inhomogeneously stretched double-stranded DNA fitted into the filament emphasizes the strategic positioning of 2 putative DNA-binding loops in a way that allows us speculate about their possibly distinct roles in nucleo-protein filament assembly and DNA strand-exchange reaction. The model suggests that the extension of a single-stranded DNA molecule upon binding of Rad51 is ensured by intercalation of Tyr-232 of the L1 loop, which might act as a docking tool, aligning protein monomers along the DNA strand upon filament assembly. Arg-235, also sitting on L1, is in the right position to make electrostatic contact with the phosphate backbone of the other DNA strand. The L2 loop position and its more ordered compact conformation makes us propose that this loop has another role, as a binding site for an incoming double-stranded DNA. Our filament structure and spectroscopic approach open the possibility of analyzing details along the multistep path of the strand-exchange reaction.

Purification and characterization of XRad51.1 protein, Xenopus RAD51 homologue: recombinant XRad51.1 promotes strand exchange reaction

Background: The RAD51 gene of Saccharomyces cerevisiae is homologous to the Escherichia coli recA gene and plays a key role in genetic recombination and DNA double‐strand break repair. To construct an improved experimental system of homologous recombination in higher eukaryotes, we have chosen the South African clawed frog, Xenopus laevis, whose egg extracts might be useful for the in vitro studies. We identified and characterized a Xenopus homologue of RAD51 gene, the XRAD51.1.

RAD51 homologues in Xenopus laevis: two distinct genes are highly expressed in ovary and testis

The RAD51 gene is a eukaryotic counterpart of the Escherichia coli recA gene which is involved in genetic recombination. Two distinct Xenopus laevis RAD51 cDNA clones (XRAD51.1 and XRAD51.2) were isolated from an oocyte cDNA library using the human RAD51 cDNA (HsRAD51) as a probe. Sequence analysis revealed that 98.2% of the amino-acid residues were identical between XRAD51.1 and XRAD51.2, and that both were 95% identical to HsRAD51. Both of the XRAD51 genes were expressed at a higher level in ovary and testis than in other somatic tissues, suggesting their involvement in meiotic recombination. The expression of XRAD51.1 was about eightfold in excess of that of XRAD51.2 in all of the tissues examined. Analysis of the rates of synonymous substitution in the coding sequences of the two XRAD51 suggests that these two genes diverged about 50 million years ago. The structural similarities of the XRAD51 proteins to RecA in E. coli and Rad51 in yeasts or vertebrates are discussed.

Purification and characterization of dihydrofolate reductase of Plasmodium falciparum expressed by a synthetic gene in Escherichia coli.

We have expressed the dihydrofolate reductase (DHFR) part of the DHFR-thymidylate synthetase complex of P. falciparum in Escherichia coli, by constructing a gene with synthetic oligonucleotides that changed the genes codon usages. The induced expression in an E. coli cell of the synthetic gene yielded a product that constituted about 30% of the total bacterial protein. The product was precipitated in an inclusion body in a cell. Its enzymatic activity was restored after denaturation and renaturation procedures with guanidine-HCl. Recombinant DHFRs with Ser or Thr at position 108 were prepared. Kinetic characterization showed that the DHFRSer108 has less of an affinity for NADPH and dihydrofolate than the DHFRThr108.

Analysis of the DNA binding site of Escherichia coli RecA protein.

To investigate the DNA binding site of RecA protein, we constructed 15 recA mutants having alterations in the regions homologous to the other ssDNA binding proteins. The in vivo analyses showed that the mutational change at Arg243, Lys248, Tyr264, or simultaneously at Lys6 and Lys19, or Lys6 and Lys23 caused severe defects in the recA functions, while other mutational changes did not. Purified RecA-K6A-K23A (Lys6 and Lys23 changed to Ala and Ala, respectively) protein was indistinguishable from the wild-type RecA protein in its binding to DNA. However, the RecA-R243A (Arg243 changed to Ala) and RecA-Y264A (Tyr264 changed to Ala) proteins were defective in binding to both ss- and ds-DNA. In self-oligomerization property, RecA-R243A was proficient but RecA-Y264A was deficient, suggesting that the RecA-R243A protein had a defect in DNA binding site and the RecA-Y264A protein was defective in its interaction with the adjacent RecA molecule. The region of residues 243-257 including the Arg243 is highly homologous to the DNA binding motif in the ssDNA binding proteins, while the eukaryotic RecA homologues have a similar structure at the amino-terminal side proximal to the nucleotide binding core. The region of residues 243-257 would be a part of the DNA binding site. The other parts of this site would be the Tyr103 and the region of residues 178-183, which were cross-linked to ssDNA. These three regions lie in a line in the crystal structure.

Arrangement of RecA protein in its active filament determined by polarized-light spectroscopy

Linear dichroism (LD) polarized-light spectroscopy is used to determine the arrangement of RecA in its large filamentous complex with DNA, active in homologous recombination. Angular orientation data for two tryptophan and seven tyrosine residues, deduced from differential LD of wild-type RecA vs. mutants that were engineered to attenuate the UV absorption of selected residues, revealed a rotation by some 40° of the RecA subunits relative to the arrangement in crystal without DNA. In addition, conformational changes are observed for tyrosine residues assigned to be involved in DNA binding and in RecA–RecA contacts, thus potentially related to the global structure of the filament and its biological function. The presented spectroscopic approach, called “Site-Specific Linear Dichroism” (SSLD), may find forceful applications also to other biologically important fibrous complexes not amenable to x-ray crystallographic or NMR structural analysis.

ADP stabilizes the human Rad51-single stranded DNA complex and promotes its DNA annealing activity

Background: Human Rad51 protein (HsRad51) is a homologue of Escherichia coli RecA protein, and involved in homologous recombination. These eukaryotic and bacterial proteins catalyse strand exchange between two homologous DNA molecules, each forming a complex with single‐stranded DNA (ssDNA) and ATP as the initial step. Both proteins hydrolyse ATP; however, the role of ATP hydrolysis appears to vary between the two proteins.