佐々 彰

The production of reactive oxygen species in response to RNA virus infection results in the oxidation of viral genomic RNA within infected cells. These oxidative RNA lesions undergo replication catalyzed by the viral replisome. G to U transversion mutations are frequently observed in the SARS-CoV-2 genome and may be linked to the replication process catalyzed by RNA-dependent RNA polymerase (RdRp) past the oxidative RNA lesion 7,8-dihydro-8-oxo-riboguanosine (8-oxo-rG). To better understand the mechanism of viral RNA mutagenesis, it is crucial to elucidate the role of RdRp in replicating across oxidative lesions. In this study, we investigated the RNA synthesis catalyzed by the reconstituted SARS-CoV-2 RdRp past a single 8-oxo-rG. The RdRp-mediated primer extension was significantly inhibited by 8-oxo-rG on the template RNA. A steady-state multiple-turnover reaction demonstrated that the turnover rate of RdRp was significantly slow when replication was blocked by 8-oxo-rG, reflecting low bypass efficiency even with prolonged reaction time. Once RdRp was able to bypass 8-oxo-rG, it preferentially incorporated rCMP, with a lesser amount of rAMP opposite 8-oxo-rG. In contrast, RdRp demonstrated greater activity in extending from the mutagenic rA:8-oxo-rG terminus compared to the lower efficiency of extension from the rC:8-oxo-rG pair. Based on steady-state kinetic analyses for the incorporation of rNMPs opposite 8-oxo-rG and chain extension from rC:8-oxo-rG or rA:8-oxo-rG, the relative bypass frequency for rA:8-oxo-rG was found to be seven-fold higher than that for rC:8-oxo-rG. Therefore, the properties of RdRp indicated in this study may contribute to the mechanism of mutagenesis of the SARS-CoV-2 genome.

Numerous chemicals are associated with carcinogenesis through epigenetic alterations in cells. To detect global epigenetic changes induced by carcinogens, the housekeeping gene can serve as a reporter locus, offering a baseline for identifying shifts in epigenetic marks. To investigate this potential, we developed a simple, cost-effective, and quantitative reporter system to assess chemically induced epigenetic effects, utilizing the thymidine kinase (TK) gene mutation assay as a foundation. Using a standard genotoxicity test cell line, human lymphoblast TK6, we edited the CpG promoter loci of the endogenous TK gene using the CRISPR/dCas9-SunTag-DNMT3A system. This epi-genotoxicity assay, employing modified mTK6 cells, provides a simple method for quantifying chemically induced epigenetic effects. The assay successfully detects both increased TK reversion rates induced by DNMT inhibitors, such as 5-Aza-2'-deoxycytidine and GSK-3484862, and, for the first time, a significant reduction in TK revertant frequency caused by the non-genotoxic carcinogen 12-O-tetradecanoylphorbol-13-acetate (TPA). Chromatin immunoprecipitation and western blotting analyses revealed that TPA treatment led to a global decrease in H3K27Ac levels, likely driven by TPA-mediated inflammation. These results demonstrate the utility of the epi-genotoxicity assay as a valuable tool for evaluating dual-directional epigenetic changes triggered by chemical exposure.

Ionizing radiation induces various types of DNA damage, and the reparability and lethal effects of DNA damage differ depending on its spatial density. Elucidating the structure of radiation-induced clustered DNA damage and its repair processes will enhance our understanding of the lethal impact of ionizing radiation and advance progress toward precise therapeutics. Previously, we developed a method to directly visualize DNA damage using atomic force microscopy (AFM) and classified clustered DNA damage into simple base damage clusters (BDCs), complex BDCs and complex double-strand breaks (DSBs). This study investigated the repair of each type of damage in DNA-repair-deficient human TK6 cells and elucidated the association between each type of clustered DNA damage and the pathway responsible for its repair postirradiation with low linear energy transfer (LET) radiation (X-rays) and high-LET radiation (Fe-ion beams) in cells. We found that base excision repair and, surprisingly, nucleotide excision repair restored simple and complex BDCs. In addition, the number of complex DSBs in wild-type cells increases 1 h postirradiation, which was most likely caused by BDC cleavage initiated with DNA glycosylases. Furthermore, complex DSBs, which are likely associated with lethality, are repaired by homologous recombination with little contribution from nonhomologous-end joining.