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Klotho is an anti-aging, single-pass transmembrane protein found mainly in the kidney. Although aging is likely to be associated with DNA damage, the involvement of Klotho in protecting cells from DNA damage is still unclear. In this study, we examined DNA damage in human kidney cells and mouse kidney tissue after ionizing radiation (IR). The depletion and overexpression of Klotho in human kidney cells reduced and increased the cell survival rates after IR, respectively. The formation of γ-H2AX foci, representing DNA damage, was significantly elevated immediately after IR in cells with Klotho depletion and decreased in cells overexpressing Klotho. These results were confirmed in mouse renal tissues after IR. Quantification of DNA damage by a comet assay revealed that the Klotho knockdown significantly increased the amount of DNA damage immediately after IR, suggesting that Klotho protects chromosomal DNA from the induction of damage, rather than facilitating DNA repair. Consistent with this notion, Klotho was detected in both the nucleus and cytoplasm. In the nucleus, Klotho may serve to protect chromosomal DNA from damage, leading to its anti-aging effects., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Japanese Biochemical Society.)

Matrin3 is a highly conserved inner nuclear matrix protein involved in multiple stages of RNA metabolism. Although Matrin3 may also play a role in DNA repair, its precise roles have remained unclear. In this study, we showed that the depletion of Matrin3 led to decreased homologous recombination (HR) efficiency and increased radiation sensitivity of cells. Matrin3-depleted cells showed impaired DNA damage-dependent focus formation of RAD51, a key protein in HR. These findings suggest that Matrin3 promotes HR by regulating RAD51., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Rim15p of the yeast Saccharomyces cerevisiae is a Greatwall-family protein kinase that inhibits alcoholic fermentation during sake brewing. To elucidate the roles of Rim15p in barley shochu fermentation, RIM15 was deleted in shochu yeast. The disruptant did not improve ethanol yield, but altered sugar and glycerol contents in the mash, suggesting that Rim15p has a novel function in carbon utilization.

Mutations of the Glu76 residue of canonical histone H2B are frequently found in cancer cells. However, it is quite mysterious how a single amino acid substitution in one of the multiple H2B genes affects cell fate. Here we found that the H2B E76K mutation, in which Glu76 is replaced by Lys (E76K), distorted the interface between H2B and H4 in the nucleosome, as revealed by the crystal structure and induced nucleosome instability in vivo and in vitro. Exogenous production of the H2B E76K mutant robustly enhanced the colony formation ability of the expressing cells, indicating that the H2B E76K mutant has the potential to promote oncogenic transformation in the presence of wild-type H2B. We found that other cancer-associated mutations of histones, H3.1 E97K and H2A.Z.1 R80C, also induced nucleosome instability. Interestingly, like the H2B E76K mutant, the H3.1 E97K mutant was minimally incorporated into chromatin in cells, but it enhanced the colony formation ability. In contrast, the H2A.Z.1 R80C mutant was incorporated into chromatin in cells, and had minor effects on the colony formation ability of the cells. These characteristics of histones with cancer-associated mutations may provide important information toward understanding how the mutations promote cancer progression.

According to an analysis of the Life Span Study cohort data conducted by the Radiation Effects Research Foundation in Hiroshima and Nagasaki, the sex-averaged excess relative risk (ERR) of all solid cancers was 0.42 Gy-Eq-1. On the other hand, analysis of the atomic bomb survivors (ABS) cohort data at Hiroshima University indicated the ERR value was 0.28 Gy-Eq-1 in Hiroshima. In both cases, initial radiation doses were derived from the dosimetry system DS02, in which the relative biological effectiveness (RBE) of neutrons was assumed to be a constant value of 10. To clarify the validity of the RBE, the authors investigated the possibility of different contributions of neutrons by using the ABS. Although there were no statistically significant differences among the estimated value of RBE (=65) and the ordinal value (=10), the corresponding ERR decreased by 30%, which might affect the interpretation of radiation health assessments.

X-rays are widely applied in the medical field for the diagnosis and treatment of diseases. Among the uses of X-rays in diagnosis, computed tomography (CT) has been established as one of the most informative diagnostic radiology examinations. Moreover, recent advances in CT scan technology have made this examination much easier and more informative and increased its application, especially in Japan. However, the radiation dose of CT scans is higher than that of simple X-ray examinations. Therefore, the health risk of a CT scan has been discussed in various studies, but is still controversial. Consequently, the biological and cytogenetic effects of CT scans are being analyzed. Here, we summarize the recent findings concerning the biological and cytogenetic effects of ionizing radiation from a CT scan, by focusing on DNA damage and chromosome aberrations.

DNA double-strand breaks (DSBs) induced by ionizing radiation (IR) are the initial and critical step in major alteration of genetic information and cell death. To prevent deleterious effects, DNA repair systems recognize and re-join DNA DSBs in human cells. It has been suggested that there are individual differences in radiosensitivity within human populations, and that variations in DNA repair genes might contribute to this heterogeneity. Because confounding factors, including age, gender, smoking, and diverse genetic backgrounds within human populations, also influence the cellular radiosensitivity, to accurately measure the effect of candidate genetic variations on radiosensitivity, it is necessary to use human cultured cells with a uniform genetic background. However, a reverse genetics approach in human cultured cells is difficult because of their low level of homologous recombination. Engineered endonucleases used in genome editing technology, however, can enable the local activation of DNA repair pathways at the human genome target site to efficiently introduce genetic variations of interest into human cultured cells. Recently, we used this technology to demonstrate that heterozygous mutations of the ATM gene, which is responsible for a hyper-radiosensitive genetic disorder, ataxia-telangiectasia, increased the number of chromosomal aberrations after IR. Thus, understanding the heterozygous mutations of radiosensitive disorders should shed light on the genetic basis underlying individual differences in radiosensitivity within human populations.

RAD52, a key factor in homologous recombination (HR), plays important roles in both RAD51-dependent and -independent HR pathways. Several studies have suggested a link between the functional regulation of RAD52 and the protein modification by a small ubiquitin-like modifier (SUMO). However, the molecular mechanism underlying the regulation of RAD52 by SUMO is unknown. To begin investigating this mechanism, we identified possible target sites for sumoylation in the human RAD52 protein by preparing a RAD52-SUMO complex using an established Escherichia coli sumoylation system. Mass spectrometry and amino acid sequencing of the enzymatically digested fragments of the purified complex revealed that the putative nuclear localization signal located near the C terminus of RAD52 was sumoylated. Biochemical studies of the RAD52-SUMO complex suggested that sumoylation at the identified site has no apparent effect on the DNA binding, D-loop formation, ssDNA annealing and RAD51-binding activities of RAD52. On the other hand, visualization of the GFP-fused RAD52 protein in the human cell that contained mutations at the identified sumoylation sites showed clear differences in the cytosolic and nuclear distributions of the protein. These results suggest the possibility of sumoylation playing an important role in the nuclear transport of RAD52.

RAD51, an essential eukaryotic DNA recombinase, promotes homologous pairing and strand exchange during homologous recombination and the recombinational repair of double strand breaks. Mutations that up- or down-regulate RAD51 gene expression have been identified in several tumors, suggesting that inappropriate expression of the RAD51 activity may cause tumorigenesis. To identify chemical compounds that affect the RAD51 activity, in the present study, we performed the RAD51-mediated strand exchange assay in the presence of 185 chemical compounds. We found that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) efficiently inhibited the RAD51-mediated strand exchange. DIDS also inhibited the RAD51-mediated homologous pairing in the absence of RPA. A surface plasmon resonance analysis revealed that DIDS directly binds to RAD51. A gel mobility shift assay showed that DIDS significantly inhibited the DNA-binding activity of RAD51. Therefore, DIDS may bind near the DNA binding site(s) of RAD51 and compete with DNA for RAD51 binding.

Bach2 is a member of the BTB-basic region leucine zipper factor family and represses transcription activity directed by the TPA response element, the Maf recognition element (MARE) and the antioxidant-responsive element. Recently, it was reported that upon oxidative stress Bach2 forms nuclear foci surrounding the promyelocytic leukaemia (PML) bodies and specifically represses the transcription around the PML bodies. Here we report that expression of the silencing mediator of retinoid and thyroid receptor (SMRT) and histone deacetylase4 (HDAC4) enhances the formation of the Bach2 foci in the nuclear matrix. SMRT mediates the HDAC4 binding to Bach2, and HDAC4 facilitates the retention of Bach2 in the foci. Scratch transcription labelling and 3D-reconstruction from the confocal images demonstrated that transcription is suppressed in and around the Bach2 foci. Indeed, Bach2 bound MARE and repressed the expression from the chromosomally integrated MARE-driven reporter gene when co-expressed with SMRT and HDAC4. Our observations suggest that both SMRT and HDAC4 play an important role in nuclear retention and the Bach2 focus formation in the mammalian cell nucleus, which may contribute to the local transcription repression.

Bach1 functions as a transcriptional repressor of heme oxygenase-1 (HO-1) and the beta-globin genes. The enhancer regions of these genes contain multiple Maf recognition elements (MAREs) to which Bach1 can bind. Previous studies have shown that increased levels of heme and cadmium induce the nuclear export of Bach1, resulting in cytoplasmic accumulation. By means of a yeast two hybrid screening using Bach1 as bait, we identified the intracellular hyaluronic acid binding protein (IHABP) as a potential regulator of Bach1. IHABP is a microtubule-associated protein that may regulate the organization of the cytoskeletal network. A series of domain analyses revealed that a region of Bach1 previously implicated in cytoplasmic accumulation was necessary for IHABP-binding. A C-terminal region of IHABP was necessary for Bach1-binding. Overexpressed Bach1 colocalized with IHABP in the cytoplasm, forming fiber-like structures on microtubules. Fluorescence recovery after photobleaching (FRAP) analysis revealed a dynamic nature of the Bach1-IHABP interaction in living cells. The repression of HO-1 reporter activity by Bach1 was attenuated by co-transfecting IHABP in a dose-dependent manner. Moreover, the overexpression of IHABP induced the endogenous HO-1 gene in NIH3T3 cells. The overall results suggest that IHABP regulates the subcelluar localization of Bach1 in order to fine-tune transactivation of Bach1 target genes such as HO-1.