Kaoru Yoshiyama

Programmed induction of endoreduplication by DNA double-strand breaks in Arabidopsis

Genome integrity is continuously threatened by external stresses and endogenous hazards such as DNA replication errors and reactive oxygen species. The DNA damage checkpoint in metazoans ensures genome integrity by delaying cell-cycle progression to repair damaged DNA or by inducing apoptosis. ATM and ATR (ataxia-telangiectasia-mutated and -Rad3-related) are sensor kinases that relay the damage signal to transducer kinases Chk1 and Chk2 and to downstream cell-cycle regulators. Plants also possess ATM and ATR orthologs but lack obvious counterparts of downstream regulators. Instead, the plant-specific transcription factor SOG1 (suppressor of gamma response 1) plays a central role in the transmission of signals from both ATM and ATR kinases. Here we show that in Arabidopsis, endoreduplication is induced by DNA double-strand breaks (DSBs), but not directly by DNA replication stress. When root or sepal cells, or undifferentiated suspension cells, were treated with DSB inducers, they displayed increased cell size and DNA ploidy. We found that the ATM–SOG1 and ATR–SOG1 pathways both transmit DSB-derived signals and that either one suffices for endocycle induction. These signaling pathways govern the expression of distinct sets of cell-cycle regulators, such as cyclin-dependent kinases and their suppressors. Our results demonstrate that Arabidopsis undergoes a programmed endoreduplicative response to DSBs, suggesting that plants have evolved a distinct strategy to sustain growth under genotoxic stress.

Suppressor of gamma response 1 (SOG1) encodes a putative transcription factor governing multiple responses to DNA damage

The Arabidopsis sog1-1 (suppressor of gamma response) mutant was originally isolated as a second-site suppressor of the radiosensitive phenotype of seeds defective in the repair endonuclease XPF. Here, we report that SOG1 encodes a putative transcription factor. This gene is a member of the NAC domain [petunia NAM (no apical meristem) and Arabidopsis ATAF1, 2 and CUC2] family (a family of proteins unique to land plants). Hundreds of genes are normally up-regulated in Arabidopsis within an hour of treatment with ionizing radiation; the induction of these genes requires the damage response protein kinase ATM, but not the related kinase ATR. Here, we find that SOG1 is also required for this transcriptional up-regulation. In contrast, the SOG1-dependent checkpoint response observed in xpf mutant seeds requires ATR, but does not require ATM. Thus, phenotype of the sog1-1 mutant mimics aspects of the phenotypes of both atr and atm mutants in Arabidopsis, suggesting that SOG1 participates in pathways governed by both of these sensor kinases. We propose that, in plants, signals related to genomic stress are processed through a single, central transcription factor, SOG1.

MUTYH-Null Mice Are Susceptible to Spontaneous and Oxidative Stress–Induced Intestinal Tumorigenesis

MUTYH is a mammalian DNA glycosylase that initiates base excision repair by excising adenine opposite 8-oxoguanine and 2-hydroxyadenine opposite guanine, thereby preventing G:C to T:A transversion caused by oxidative stress. Recently, biallelic germ-line mutations of MUTYH have been found in patients predisposed to a recessive form of hereditary multiple colorectal adenoma and carcinoma with an increased incidence of G:C to T:A somatic mutations in the APC gene. In the present study, a systematic histologic examination revealed that more spontaneous tumors had developed in MUTYH-null mice (72 of 121; 59.5%) than in the wild type (38 of 109; 34.9%). The increased incidence of intestinal tumors in MUTYH-null mice (11 tumors in 10 of 121 mice) was statistically significant compared with the wild type (no intestinal tumors in 109 mice). Two adenomas and seven adenocarcinomas were observed in the small intestines, and two adenomas but no carcinomas were found in the colons. In MUTYH-null mice treated with KBrO(3), the occurrence of small intestinal tumors dramatically increased. The mean number of polyps induced in the small intestines of these mice was 61.88 (males, 72.75; females, 51.00), whereas it was 0.85 (males, 0.50; females, 1.00) in wild-type mice. The tumors developed predominantly in the duodenum and in the upper region of the (jejunum) small intestines. We conclude that MUTYH suppresses spontaneous tumorigenesis in mammals, thus providing experimental evidence for the association between biallelic germ-line MUTYH mutations and a recessive form of human hereditary colorectal adenoma and carcinoma.

The Arabidopsis SIAMESE-RELATED Cyclin-Dependent Kinase Inhibitors SMR5 and SMR7 Regulate the DNA Damage Checkpoint in Response to Reactive Oxygen Species

Reactive oxygen species (ROS) cause DNA damage. In this work, two SIAMESE/SIAMESE-RELATED (SIM/SMR) genes that encode cyclin-dependent kinase inhibitors are described as being part of a signaling pathway that arrests cell proliferation in response to ROS, revealing a novel cell cycle checkpoint-signaling cascade. Whereas our knowledge about the diverse pathways aiding DNA repair upon genome damage is steadily increasing, little is known about the molecular players that adjust the plant cell cycle in response to DNA stress. By a meta-analysis of DNA stress microarray data sets, three family members of the SIAMESE/SIAMESE-RELATED (SIM/SMR) class of cyclin-dependent kinase inhibitors were discovered that react strongly to genotoxicity. Transcriptional reporter constructs corroborated specific and strong activation of the three SIM/SMR genes in the meristems upon DNA stress, whereas overexpression analysis confirmed their cell cycle inhibitory potential. In agreement with being checkpoint regulators, SMR5 and SMR7 knockout plants displayed an impaired checkpoint in leaf cells upon treatment with the replication inhibitory drug hydroxyurea (HU). Surprisingly, HU-induced SMR5/SMR7 expression depends on ATAXIA TELANGIECTASIA MUTATED (ATM) and SUPPRESSOR OF GAMMA RESPONSE1, rather than on the anticipated replication stress-activated ATM AND RAD3-RELATED kinase. This apparent discrepancy was explained by demonstrating that, in addition to its effect on replication, HU triggers the formation of reactive oxygen species (ROS). ROS-dependent transcriptional activation of the SMR genes was confirmed by different ROS-inducing conditions, including high-light treatment. We conclude that the identified SMR genes are part of a signaling cascade that induces a cell cycle checkpoint in response to ROS-induced DNA damage.

Impact of reactive oxygen species on spontaneous mutagenesis in Escherichia coli

Reactive oxygen species (ROS) are potent oxidants that attack chromosomal DNA and free nucleotides, leading to oxidative DNA damage that causes genetic alterations. To avoid the ROS‐mediated mutagenesis, cells have elaborate mechanisms including powerful antioxidant components and repair pathways that eliminate oxidative DNA damage. Because of the effective anti‐mutagenic functions, it has been unclear to what extent the ROS contribute to spontaneous mutagenesis. Here we show that a significant portion of spontaneous mutations is actually caused by the ROS in aerobically growing Escherichia coli cells. Using the rpsL gene as a mutational target sequence, we established an experimental procedure to analyze spontaneous mutations occurring under a strictly anaerobic condition. Strong mutator phenotypes of cells defective in both mutM and mutY genes or ones lacking mutT gene were completely suppressed under the anaerobic condition, indicative of an absence of hydroxyl radicals in the cells. From a series of analyses with wild‐type E. coli cells grown under different redox conditions, it appeared that 89% of base substitutions were caused by the ROS, especially hydroxyl radicals, in cells growing in the atmosphere. The ROS‐mediated spontaneous mutations included highly site‐specific base substitutions, two types of randomly occurring transversions, G:C→C:G and A:T→T:A, and –1 frameshifts at non‐iterated base sequences.

ATM‐mediated phosphorylation of SOG1 is essential for the DNA damage response in Arabidopsis

Arabidopsis SOG1 (suppressor of gamma response 1) is a plant‐specific transcription factor that governs the DNA damage response. Here we report that SOG1 is phosphorylated in response to DNA damage, and that this phosphorylation is mediated by the sensor kinase ataxia telangiectasia mutated (ATM). We show that SOG1 phosphorylation is crucial for the response to DNA damage, including transcriptional induction of downstream genes, transient arrest of cell division and programmed cell death. Although the amino‐acid sequences of SOG1 and the mammalian tumour suppressor p53 show no similarity, this study demonstrates that ATM‐mediated phosphorylation of a transcription factor has a pivotal role in the DNA damage response in both plants and mammals.

DNA Damage Response in Plants: Conserved and Variable Response Compared to Animals

The genome of an organism is under constant attack from endogenous and exogenous DNA damaging factors, such as reactive radicals, radiation, and genotoxins. Therefore, DNA damage response systems to sense DNA damage, arrest cell cycle, repair DNA lesions, and/or induce programmed cell death are crucial for maintenance of genomic integrity and survival of the organism. Genome sequences revealed that, although plants possess many of the DNA damage response factors that are present in the animal systems, they are missing some of the important regulators, such as the p53 tumor suppressor. These observations suggest differences in the DNA damage response mechanisms between plants and animals. In this review the DNA damage responses in plants and animals are compared and contrasted. In addition, the function of SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a plant-specific transcription factor that governs the robust response to DNA damage, is discussed.

SOG1: a master regulator of the DNA damage response in plants

The DNA damage response (DDR) is a critical mechanism to maintain the genome stability of an organism upon exposure to endogenous and exogenous DNA-damaging factors. The DDR system is particularly important for plants as these organisms, owing to their intrinsic immobility, are inevitably exposed to environmental stress factors, some of which induce DNA damage. Arabidopsis thaliana has orthologs of several DDR factors that are present in animals; however, some of the important animal regulators, such as the tumor suppressor p53 and the DDR kinases CHK1 and CHK2, have not been found in plants. These observations imply a unique DDR system in plants. The present review focuses on recent advances in our understanding of the DDR in A. thaliana and, in particular, on the function and role of SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a plant-specific transcription factor that regulates the DDR. The most obvious response to DNA damage in A. thaliana is a rapid and robust change in the transcriptional regulation of numerous genes, in which SOG1 is an essential regulatory factor. Mutation of SOG1 causes various defects in the activation of cell cycle arrest, programmed cell death, and endoreduplication in response to DNA damage. These observations indicate that SOG1 is a master regulator of the DDR. Phylogenetic analyses of SOG1 reveal that orthologs of this crucial transcription factor are present not only in angiosperms but also in gymnosperms, suggesting that the SOG1 system is conserved across spermatophytes. Finally, future prospects for SOG1 research are also discussed.

Spontaneous mutagenesis associated with nucleotide excision repair in Escherichia coli

The vast majority of spontaneous mutations occurring in Escherichia coli are thought to be derived from spontaneous DNA lesions, which include oxidative base damage. Systems for removing intrinsic mutagens and repairing DNA lesions contribute to the suppression of spontaneous mutations. Nucleotide excision repair (NER) is a general DNA repair system that eliminates various kinds of lesions from DNA. We therefore predicted that NER might be involved in suppression of spontaneous mutations, and analyzed base substitutions occurring spontaneously within the rpoB gene in NER‐proficient (wild‐type), ‐deficient and ‐overproducing E. coli strains. Surprisingly, the mutation frequency was lower in NER‐deficient strains, and higher in NER‐overproducing strains, than in the NER‐proficient strain. These results suggest, paradoxically, that NER contributes to the generation of spontaneous mutation rather than to its suppression under normal growth conditions, and that transcription‐coupled repair also participates in this process. Using E. coli strains that carried an editing exonuclease‐deficient polA mutation, we further obtained data suggesting that unnecessary NER might account for these findings, so that errors introduced during repair DNA synthesis by DNA polymerase I would result in unwanted base substitutions. The repair system itself may thus be an important generator of spontaneous mutation.

Spontaneous Hotspot Mutations Resistant to Mismatch Correction in Escherichia coli: Transcription-dependent Mutagenesis Involving Template-switching Mechanisms

The generation and stabilization of spontaneous mutations are affected by many factors, including the accuracy of DNA replication, the generation of spontaneous DNA lesions, and the capacity of mutation-avoidance systems. However, little is known about the causes of spontaneous mutations in cells with fully active mutation-avoidance systems. Using the rpsL forward mutation assay, we previously found that the directionality of replication fork movement significantly affects spontaneous mutagenesis in Escherichia coli. In particular, sequence substitutions and a hotspot type of single-base frameshift, both of which are caused by quasipalindrome-directed mutagenesis, appeared to depend on the directionality of the replication fork. These mutations are also resistant to post-replicative mismatch correction. Here, we show that the level of transcription of the rpsL gene strongly affects spontaneous mutagenesis at two mutational hotspot sites in the target sequence, one for a T-->G base substitution and the other for a+1 single-base frameshift. Mutation frequencies at the hotspot sites were below a detectable level when the transcription of the target sequence was tightly suppressed, but were dramatically increased when the target sequence was highly transcribed. Both of the hotspot mutations were also dependent on the directionality of the replication fork and were caused by quasipalindrome-directed mutagenesis. The frequencies of the hotspot mutations were unchanged in a mismatch-repair deficient strain, indicating that the hotspot mutations are resistant to the mismatch correction. Based on these findings, we propose a novel mutagenic process for these hotspot mutations that depends on transcription and involves template-switching mechanisms induced by spontaneous DNA lesions.