Hisaji Maki

Fate of DNA replication fork encountering a single DNA lesion during oriC plasmid DNA replication in vitro

Background: The inhibition of DNA replication fork progression by DNA lesions can lead to cell death or genome instability. However, little is known about how such DNA lesions affect the concurrent synthesis of leading‐ and lagging‐strand DNA catalysed by the protein machinery used in chromosomal replication. Using a system of semi‐bidirectional DNA replication of an oriC plasmid that employs purified replicative enzymes and a replication‐terminating protein of Escherichia coli, we examined the dynamics of the replication fork when it encounters a single abasic DNA lesion on the template DNA.

Mutational specificity of mice defective in the MTH1 and/or the MSH2 genes.

Oxidative damage of nucleotides within DNA or precursor pools caused by oxygen radicals is thought to play an important role in spontaneous mutagenesis, as well as carcinogenesis and aging. In particular, 8-oxodGTP and 2-OHdATP are potent mutagenic substrate for DNA synthesis. Mammalian MTH1 catalyzes hydrolysis of these mutagenic substrates, suggesting that it functions to prevent mutagenesis caused by these oxidized nucleotides. We have established MTH1(-/-) mice lacking the 8-oxodGTPase activity, which were shown to be susceptible to lung, liver and stomach cancers. To examine in vivo mutation events due to the MTH1-deficiency, a reporter gene, rpsL of Escherichia coli, was introduced into MTH1(-/-) mice. Interestingly, the net frequency of rpsL(-) forward mutants showed no apparent increase in MTH1(-/-) mice as compared to MTH1(+/+) mice. However, we found differences between these two genotypes in the class- and site-distributions of the rpsL(-) mutations recovered from the mice. Unlike MutT-deficient E. coli showing 1000-fold higher frequency of A:T-->C:G transversion than the wild type cells, an increase in frequency of A:T-->C:G transversion was not evident in MTH1 nullizygous mice. Nevertheless, the frequency of single-base frameshifts at mononucleotide runs was 5.7-fold higher in spleens of MTH1(-/-) mice than in those of wild type mice. Since the elevated incidence of single-base frameshifts at mononucleotide runs is a hallmark of the defect in MSH2-dependent mismatch repair system, this weak site-specific mutator effect of MTH1(-/-) mice could be attributed to a partial sequestration of the mismatch repair function that may act to correct mispairs with the oxidized nucleotides. Consistent with this hypothesis, a significant increase in the frequency of G:C-->T:A transversions was observed with MTH1(-/-) MSH2(-/-) mice over MSH2(-/-) mice alone. These results suggest a possible involvement of multiple anti-mutagenic pathways, including the MTH1 protein and other repair system(s), in mutagenesis caused by the oxidized nucleotides.

Involvement of RAD9-Dependent Damage Checkpoint Control in Arrest of Cell Cycle, Induction of Cell Death, and Chromosome Instability Caused by Defects in Origin Recognition Complex in Saccharomyces cerevisiae

ABSTRACT Perturbation of origin firing in chromosome replication is a possible cause of spontaneous chromosome instability in multireplicon organisms. Here, we show that chromosomal abnormalities, including aneuploidy and chromosome rearrangement, were significantly increased in yeast diploid cells with defects in the origin recognition complex. The cell cycle of orc1-4/orc1-4 temperature-sensitive mutant was arrested at the G2/M boundary, after several rounds of cell division at the restrictive temperature. However, prolonged incubation of the mutant cells at 37°C led to abrogation of G2 arrest, and simultaneously the cells started to lose viability. A sharp increase in chromosome instability followed the abrogation of G2 arrest. In orc1-4/orc1-4 rad9Δ/rad9Δ diploid cells grown at 37°C, G2 arrest and induction of cell death were suppressed, while chromosome instability was synergistically augmented. These findings indicated that DNA lesions caused by a defect in Orc1p function trigger the RAD9-dependent checkpoint control, which ensures genomic integrity either by stopping the cell cycle progress until lesion repair, or by inducing cell death when the lesion is not properly repaired. At semirestrictive temperatures, orc2-1/orc2-1 diploid cells demonstrated G2 arrest and loss of cell viability, both of which require RAD9-dependent checkpoint control. However, chromosome instability was not induced in orc2-1/orc2-1 cells, even in the absence of the checkpoint control. These data suggest that once cells lose the damage checkpoint control, perturbation of origin firing can be tolerated by the cells. Furthermore, although a reduction in origin-firing capacity does not necessarily initiate chromosome instability, the Orc1p possesses a unique function, the loss of which induces instability in the chromosome.

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.

Spontaneous tumorigenesis and mutagenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase

Oxygen radicals, which can be produced through normal cellular metabolism as well as X-ray irradiation, are thought to play an important role in mutagenesis and tumorigenesis. Among various classes of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is most important because of its abundance and mutagenicity. The MTH1 gene encodes an enzyme that hydrolyzes 8-oxo-dGTP to monophosphate in the nucleotide pool, thereby preventing occurrence of mutations. When examined 18 months after birth, a greater number of tumors had formed in the lungs, livers and stomachs of MTH1-deficient mice, as compared with wild-type mice. Next, we analysed the mutation frequency and their spectra in each MTH1-deficient or proficient mice at the age of 4 and 24 weeks. The mutation frequency on the rpsL transgene in the spleen samples was determined. However, the spontaneous mutation frequency observed in the spleen samples from the MTH1−/− mice showed no apparent increase compared to the value of the one in the MTH1+/+ mice. Furthermore, the site distribution of the mutations that occurred on the rpsL gene was slightly different between these two MTH1 genotypes.