Takehiko Shibata

Distinct roles of two separable in vitro activities of yeast Mre11 in mitotic and meiotic recombination

In Saccharomyces cerevisiae, Mre11 protein is involved in both double‐strand DNA break (DSB) repair and meiotic DSB formation. Here, we report the correlation of nuclease and DNA‐binding activities of Mre11 with its functions in DNA repair and meiotic DSB formation. Purified Mre11 bound to DNA efficiently and was shown to have Mn2+‐dependent nuclease activities. A point mutation in the N‐terminal phosphoesterase motif (Mre11D16A) resulted in the abolition of nuclease activities but had no significant effect on DNA binding. The wild‐type level of nuclease activity was detected in a C‐terminal truncated protein (Mre11ΔC49), although it had reduced DNA‐binding activity. Phenotypes of the corresponding mutations were also analyzed. The mre11D16A mutation conferred methyl methanesulfonate‐sensitivity to mitotic cells and caused the accumulation of unprocessed meiotic DSBs. The mre11ΔC49 mutant exhibited almost wild‐type phenotypes in mitosis. However, in meiosis, no DSB formation could be detected and an aberrant chromatin configuration was observed at DSB sites in the mre11ΔC49 mutant. These results indicate that Mre11 has two separable functional domains: the N‐terminal nuclease domain required for DSB repair, and the C‐terminal dsDNA‐binding domain essential to its meiotic functions such as chromatin modification and DSB formation.

Stepwise chromatin remodelling by a cascade of transcription initiation of non-coding RNAs

Recent transcriptome analyses using high-density tiling arrays and data from large-scale analyses of full-length complementary DNA libraries by the FANTOM3 consortium demonstrate that many transcripts are non-coding RNAs (ncRNAs). These transcriptome analyses indicate that many of the non-coding regions, previously thought to be functionally inert, are actually transcriptionally active regions with various features. Furthermore, most relatively large (∼several kilobases) polyadenylated messenger RNA transcripts are transcribed from regions harbouring little coding potential. However, the function of such ncRNAs is mostly unknown and has been a matter of debate. Here we show that RNA polymerase II (RNAPII) transcription of ncRNAs is required for chromatin remodelling at the fission yeast Schizosaccharomyces pombe fbp1+ locus during transcriptional activation. The chromatin at fbp1+ is progressively converted to an open configuration, as several species of ncRNAs are transcribed through fbp1+. This is coupled with the translocation of RNAPII through the region upstream of the eventual fbp1+ transcriptional start site. Insertion of a transcription terminator into this upstream region abolishes both the cascade of transcription of ncRNAs and the progressive chromatin alteration. Our results demonstrate that transcription through the promoter region is required to make DNA sequences accessible to transcriptional activators and to RNAPII.

Meiotic association between Spo11 regulated by Rec102, Rec104 and Rec114

Meiotic recombination is initiated by DNA double-stranded break (DSB) formation catalyzed by Spo11, a type-II topoisomerase-like transesterificase, presumably via a dimerization-mediated mechanism. We demonstrate the existence of in vivo interactions between Spo11 proteins carrying distinct tags, and the chromatin-binding and DSB activity of tagged Spo11 at innate and targeted DSB sites upon fusion to the Gal4 DNA-binding domain. First we identified the interaction between Spo11-3FLAG and Gal4BD-Spo11 proteins, and established that this interaction specifically occurs at the time of DSB formation. We then observed that presence of the Gal4BD-spo11Y135F (nuclease-deficient) protein allows Spo11-3FLAG recruitment at the GAL2 locus, indicative of the formation of a hetero-complex near the GAL2 UAS sites, but no formation of double- or single-strand breaks. Spo11 self-interaction around the GAL2 DSB site depends on other proteins for DSB formation, in particular Rec102, Rec104 and Rec114. Together, these results suggest that in vivo self-association of Spo11 during meiosis is genetically regulated. The results are discussed in relation to possible roles of Spo11 self-interaction in the control of the cleavage activity.

Repair of ultraviolet-induced DNA damage in the subcellular systems of Bacillus subtilis

Abstract Repair of UV-induced lesions in DNA was studied with various kinds of subcellular system prepared from Bacillus subtilis . The degree of repair during the post-irradiation incubation period was calculated from marker survivals in transforming DNA. Systems consisting mainly of non-viable spherical cells and subcellular fragments, as well as systems consisting of colony-forming protoplasts, were able to repair UV-induced lesions as efficiently as intact cell systems. A Teflon homogenate, a freeze-and-thawed product and an osmotic shockate were also examined. The former two systems showed high repair activity, but the last did not. Attempts to repair the lesions with a supernatant fraction of Teflon homogenate were unsuccessful. In contrast with the active protoplast derivatives, toluene-treated cells were inert with respect to repair even when supplemented with substrates and cofactors although they retained DNA-synthesizing activity under similar conditions.

Single-stranded DNA catenation mediated by human EVL and a type I topoisomerase

The human Ena/Vasp-like (EVL) protein is considered to be a bifunctional protein, involved in both actin remodeling and homologous recombination. In the present study, we found that human EVL forms heat-stable multimers of circular single-stranded DNA (ssDNA) molecules in the presence of a type I topoisomerase in vitro. An electron microscopic analysis revealed that the heat-stable ssDNA multimers formed by EVL and topoisomerase were ssDNA catemers. The ssDNA catenation did not occur when either EVL or topoisomerase was omitted from the reaction mixture. A deletion analysis revealed that the ssDNA catenation completely depended on the annealing activity of EVL. Human EVL was captured from a human cell extract by TOPO IIIα-conjugated beads, and the interaction between EVL and TOPO IIIα was confirmed by a surface plasmon resonance analysis. Purified TOPO IIIα catalyzed the ssDNA catenation with EVL as efficiently as the Escherichia coli topoisomerase I. Since the ssDNA cutting and rejoining reactions, which are the sub-steps of ssDNA catenation, may be an essential process in homologous recombination, EVL and TOPO IIIα may function in the processing of DNA intermediates formed during homologous recombination.