Masahiko S. Satoh

Double-stranded DNA binding domain of poly(ADP-ribose) polymerase-1 and molecular insight into the regulation of its activity.

Poly(ADP-ribose) polymerase-1 (PARP-1) modifies various proteins, including itself, with ADP-ribose polymers (automodification). Polymer synthesis is triggered by binding of its zinc finger 1 (Zn1) and 2 (Zn2) to DNA breaks and is followed by inactivation through automodification. The multiple functional domains of PARP-1 appear to regulate activation and automodification-mediated inactivation of PARP-1. However, the roles of these domains in activation-inactivation processes are not well understood. Our results suggest that Zn1, Zn2, and a domain identified in this study, the double-stranded DNA binding (DsDB) domain, are involved in DNA break-dependent activation of PARP-1. We found that binding of the DsDB domain to double-stranded DNA and DNA break recognition by Zn1 and Zn2, whose actual binding targets are likely to be single-stranded DNA, lead to the activation of PARP-1. In turn, the displacement of single- and double-stranded DNA from Zn2 and the DsDB domain caused by ADP-ribose polymer synthesis results in the dissociation of PARP-1 from DNA breaks and thus its inactivation. We also found that the WGR domain is one of the domains involved in the RNA-dependent activation of PARP-1. Furthermore, because zinc finger 3 (Zn3) has the ability to bind to single-stranded RNA, it may have an indirect role in RNA-dependent activation. PARP-1 functional domains, which are involved in oligonucleic acid binding, therefore coordinately regulate PARP-1 activity depending on the status of the neighboring oligonucleic acids. Based on these results, we proposed a model for the regulation of PARP-1 activity.

Single-cell lineage tracking analysis reveals that an established cell line comprises putative cancer stem cells and their heterogeneous progeny.

Mammalian cell culture has been used in many biological studies on the assumption that a cell line comprises putatively homogeneous clonal cells, thereby sharing similar phenotypic features. This fundamental assumption has not yet been fully tested; therefore, we developed a method for the chronological analysis of individual HeLa cells. The analysis was performed by live cell imaging, tracking of every single cell recorded on imaging videos, and determining the fates of individual cells. We found that cell fate varied significantly, indicating that, in contrast to the assumption, the HeLa cell line is composed of highly heterogeneous cells. Furthermore, our results reveal that only a limited number of cells are immortal and renew themselves, giving rise to the remaining cells. These cells have reduced reproductive ability, creating a functionally heterogeneous cell population. Hence, the HeLa cell line is maintained by the limited number of immortal cells, which could be putative cancer stem cells.

Induction of Base Damages Representing a High Risk Site for Double-strand DNA Break Formation in Genomic DNA by Exposure of Cells to DNA Damaging Agents

DNA repair is known as a defense mechanism against genotoxic insults. However, the most lethal type of DNA damages, double-strand DNA breaks (DSBs), can be produced by DNA repair. We have previously demonstrated that when long patch base excision repair attempts to repair a synthetic substrate containing two uracils, the repair produces DSBs (Vispe, S. and Satoh, M. S. (2000) J. Biol. Chem. 275, 27386-27392 and Vispe, S., Ho, E. L., Yung, T. M., and Satoh, M. S. (2003) J. Biol. Chem. 278, 35279-35285). In this synthetic substrate, the two uracils are located on the opposite DNA strands (separated by an intervening sequence stable at 37 °C) and represent a high risk site for DSB formation. It is not clear, however, whether similar high risk sites are also induced in genomic DNA by exposure to DNA damaging agents. Thus, to investigate the mechanisms of DSB formation, we have modified the DSB formation assay developed previously and demonstrated that high risk sites for DSB formation are indeed generated in genomic DNA by exposure of cells to alkylating agents. In fact, genomic DNA containing alkylated base damages, which could represent high risk sites, are converted into DSBs by enzymes present in extracts prepared from cells derived from clinically normal individuals. Furthermore, DSBs are also produced by extracts from cells derived from ataxia-telangiectasia patients who show cancer proneness due to an impaired response to DSBs. These results suggest the presence of a novel link between base damage formation and DSBs and between long patch base excision repair and human diseases that occur due to an impaired response to DSB.

Basal level of p53 regulates cell population homeostasis

p53 regulates a broad range of processes in cells exposed to various stresses. Although, in unstressed cells, p53 is maintained at low levels, it is unclear if these low levels of p53 have any functional roles. Thus, we investigated the roles of basal levels of p53 using a single-cell lineage tracking technique. We compared the spatiotemporal responses of p53-proficient cancer cells with cells in which p53 gene expression had been silenced. We found that the p53-proficient cell population included a subpopulation of fast-growing cells with a shorter cell-doubling time than other slow-growing cells. p53 silencing reduced the cell-doubling time of the slow-growing cells, thus converting them to faster growing cells. In contrast, the growth rate of the fast-growing cells was unaffected by basal levels of p53, suggesting that growth of slow-growing but not fast-growing cells were regulated by p53 levels. In addition, silencing p53 increased the incidences of multipolar cell division and cell death among the slow-growing cells. These results suggest that basal levels of p53 act to maintain a balance between the fast- and slow-growing cell subpopulations and to suppress the occurrence of abnormal cellular events. We therefore propose that basal levels of p53 help to control cell population homeostasis by regulating the growth and maintaining the integrity of slow-growing cells.

Galectin-3 and N-acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy

The muscle membrane, sarcolemma, must be firmly attached to the basal lamina. The failure of proper attachment results in muscle injury, which is the underlying cause of Duchenne muscular dystrophy (DMD), in which mutations in the dystrophin gene disrupts the firm adhesion. In patients with DMD, even moderate contraction causes damage, leading to progressive muscle degeneration. The damaged muscles are repaired through myogenesis. Consequently, myogenesis is highly active in patients with DMD, and the repeated activation of myogenesis leads to the exhaustion of the myogenic stem cells. Therefore, approaches to reducing the risk of the exhaustion are to develop a treatment that strengthens the interaction between the sarcolemma and the basal lamina and increases the efficiency of the myogenesis. Galectin‐3 is an oligosaccharide‐binding protein and is known to be involved in cell–cell interactions and cell–matrix interactions. Galectin‐3 is expressed in myoblasts and skeletal muscle, although its function in muscle remains elusive. In this study, we found evidence that galectin‐3 and the monosaccharide N‐acetylglucosamine, which increases the synthesis of binding partners (oligosaccharides) of galectin‐3, promote myogenesis in vitro. Moreover, in the mdx mouse model of DMD, treatment with N‐acetylglucosamine increased muscle‐force production. The results suggest that treatment with N‐acetylglucosamine might mitigate the burden of DMD.—Rancourt, A., Dufresne, S. S., St‐Pierre, G., Levesque, J.‐C., Nakamura, H., Kikuchi, Y., Satoh, M. S., Frenette, J., Sato, S. Galectin‐3 and N‐acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy. FASEB J. 32, 6445–6455 (2018). www.fasebj.org