Naoko Shiomi

Postnatal Growth Failure, Short Life Span, and Early Onset of Cellular Senescence and Subsequent Immortalization in Mice Lacking the Xeroderma Pigmentosum Group G Gene

ABSTRACT The xeroderma pigmentosum group G (XP-G) gene (XPG) encodes a structure-specific DNA endonuclease that functions in nucleotide excision repair (NER). XP-G patients show various symptoms, ranging from mild cutaneous abnormalities to severe dermatological impairments. In some cases, patients exhibit growth failure and life-shortening and neurological dysfunctions, which are characteristics of Cockayne syndrome (CS). The known XPG protein function as the 3′ nuclease in NER, however, cannot explain the development of CS in certain XP-G patients. To gain an insight into the functions of the XPG protein, we have generated and examined mice lacking xpg (the mouse counterpart of the humanXPG gene) alleles. The xpg-deficient mice exhibited postnatal growth failure and underwent premature death. SinceXPA-deficient mice, which are totally defective in NER, do not show such symptoms, our data indicate that XPG performs an additional function(s) besides its role in NER. Our in vitro studies showed that primary embryonic fibroblasts isolated from thexpg-deficient mice underwent premature senescence and exhibited the early onset of immortalization and accumulation of p53.

Human RAD18 is involved in S phase-specific single-strand break repair without PCNA monoubiquitination

Switching from a replicative to a translesion polymerase is an important step to further continue on replication at the site of DNA lesion. Recently, RAD18 (a ubiquitin ligase) was shown to monoubiquitinate proliferating cell nuclear antigen (PCNA) in cooperation with RAD6 (a ubiquitin-conjugating enzyme) at the replication-stalled sites, causing the polymerase switch. Analyzing RAD18-knockout (RAD18−/−) cells generated from human HCT116 cells, in addition to the polymerase switch, we found a new function of RAD18 for S phase-specific DNA single-strand break repair (SSBR). Unlike the case with polymerase switching, PCNA monoubiquitination was not necessary for the SSBR. When compared with wild-type HCT116 cells, RAD18−/− cells, defective in the repair of X-ray-induced chromosomal aberrations, were significantly hypersensitive to X-ray-irradiation and also to the topoisomerase I inhibitor camptothecin (CPT) capable of inducing single-strand breaks but were not so sensitive to the topoisomerase II inhibitor etoposide capable of inducing double-strand breaks. However, such hypersensitivity to CPT observed with RAD18−/− cells was limited to only the S phase due to the absence of the RAD18 S phase-specific function. Furthermore, the defective SSBR observed in S phase of RAD18−/− cells was also demonstrated by alkaline comet assay.

Identification of the XPG Region That Causes the Onset of Cockayne Syndrome by Using Xpg Mutant Mice Generated by the cDNA-Mediated Knock-In Method

ABSTRACT In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause early onset of Cockayne syndrome (CS) in some patients (XPG/CS). The CS-causing mutations in such patients all produce truncated XPG proteins. To test the hypothesis that the CS phenotype, with characteristics such as growth retardation and a short life span in XPG/CS patients, results from C-terminal truncations, we constructed mutants with C-terminal truncations in mouse XPG (Xpg) (from residue D811 to the stop codon [XpgD811stop] and deletion of exon 15 [XpgΔex15]). In the XpgD811stop and XpgΔex15 mutations, the last 360 and 183 amino acids of the protein were deleted, respectively. To generate Xpg mutant mice, we devised the shortcut knock-in method by replacing genomic DNA with a mutated cDNA fragment (cDNA-mediated knock in). The control mice, in which one-half of Xpg genomic DNA fragment was replaced with a normal Xpg cDNA fragment, had a normal growth rate, a normal life span, normal sensitivity to UV light, and normal DNA repair ability, indicating that the Xpg gene partially replaced with the normal cDNA fragment retained normal functions. The XpgD811stop homozygous mice exhibited growth retardation and a short life span, but the XpgΔex15 homozygous mice did not, indicating that deletion of the last 360 amino acids results in the CS phenotype but deletion of the last 183 amino acids does not. The XpgD811stop homozygous mice, however, exhibited a slightly milder CS phenotype than did the Xpg null mutant mice, indicating that the XpgD811stop protein still retains some Xpg function that affects the severity of the CS phenotype.

An ERCC5 gene with homology to yeast RAD2 is involved in group G xeroderma pigmentosum

We have isolated a human excision repair gene ERCC5 which complements the defect of the mouse UV-sensitive mutant XL216 (rodent complementation group 5). Here we report cDNA cloning of human and mouse ERCC5 genes using an exon containing an ERCC5 fragment as a probe. The ERCC5 cDNA encodes a predicted 133-kDa nuclear protein that shares some homology with the product of the yeast DNA repair gene RAD2. Transfection with mouse ERCC5 cDNA restored normal levels of UV resistance to XL216 cells. Microinjection of ERCC5 cDNA specifically restored the defect of xeroderma pigmentosum group G cells (XP-G) as measured by unscheduled DNA synthesis, and XP-G cells stably transformed with ERCC5 cDNA showed nearly normal UV resistance.

Purkinje cell degeneration in mice lacking the xeroderma pigmentosum group G gene

Laboratory mice carrying the nonfunctional xeroderma pigmentosum group G gene (the mouse counterpart of the human XPG gene) alleles have been generated by using gene‐targeting and embryonic stem cell technology. Homozygote animals of this autosomal recessive disease exhibited signs and symptoms, such as postnatal growth retardation, reduced levels of activity, progressive ataxia and premature death, similar to the clinical manifestations of Cockayne syndrome (CS). Histological analysis of the cerebellum revealed multiple pyknotic cells in the Purkinje cell layer of the xpg homozygotes, which had atrophic cell bodies and shrunken nuclei. Further examination by an immunohistochemistry for calbindin‐D 28k (CaBP) showed that a large number of immunoreactive Purkinje cells were atrophic and their dendritic trees were smaller and shorter than in wild‐type littermates. These results indicated a marked degeneration of Purkinje cells in the xpg mutant cerebellum. Study by in situ detection of DNA fragmentation in the cerebellar cortex demonstrated that some deoxynucleotidyl transferase (TdT)‐mediated dUTP‐biotin in situ nick labeling (TUNEL)‐positive cells appeared in the granule layer of the mutant mice, but few cell deaths were confirmed in the Purkinje layer. These results suggested Purkinje cell degeneration in the mutant cerebellum was underway, in which much Purkinje cell death had not appeared, and the appearance of some abnormal cerebellar symptoms in the xpg‐deficient mice was not only due to a marked Purkinje cell degeneration, but also to damage of other cells. J. Neurosci. Res. 64:348–354, 2001.

SPICE-NIRS microbeam: a focused vertical system for proton irradiation of a single cell for radiobiological research.

The Single Particle Irradiation system to Cell (SPICE) facility at the National Institute of Radiological Sciences (NIRS) is a focused vertical microbeam system designed to irradiate the nuclei of adhesive mammalian cells with a defined number of 3.4 MeV protons. The approximately 2-μm diameter proton beam is focused with a magnetic quadrupole triplet lens and traverses the cells contained in dishes from bottom to top. All procedures for irradiation, such as cell image capturing, cell recognition and position calculation, are automated. The most distinctive characteristic of the system is its stability and high throughput; i.e. 3000 cells in a 5 mm × 5 mm area in a single dish can be routinely irradiated by the 2-μm beam within 15 min (the maximum irradiation speed is 400 cells/min). The number of protons can be set as low as one, at a precision measured by CR-39 detectors to be 99.0%. A variety of targeting modes such as fractional population targeting mode, multi-position targeting mode for nucleus irradiation and cytoplasm targeting mode are available. As an example of multi-position targeting irradiation of mammalian cells, five fluorescent spots in a cell nucleus were demonstrated using the γ-H2AX immune-staining technique. The SPICE performance modes described in this paper are in routine use. SPICE is a joint-use research facility of NIRS and its beam times are distributed for collaborative research.

Rodent complementation group 8 (ERCC8) corresponds to Cockayne syndrome complementation group A.

US31 is a UV-sensitive mutant cell line (rodent complementation group 8) derived from a mouse T cell line L5178Y. We analyzed removal kinetics for UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts in US31 cells using monoclonal antibodies against these photoproducts. While nearly all (6-4) photoproducts were repaired within 6 h after UV-irradiation, more than 70% of cyclobutane pyrimidine dimers remained unrepaired even 24 h after UV-irradiation. These kinetics resembled those of Cockayne syndrome (CS) cells. Since US31 cells had a low efficiency of cell fusion and transfection, which hampered both complementation tests and gene cloning, we constructed fibroblastic complementation group 8 cell line 6L1030 by fusion of US31 cells with X-irradiated normal mouse fibroblastic LTA cells. Complementation tests by cell fusion and transfection using 6L1030 cells revealed that rodent complementation group 8 corresponded to CS complementation group A.

Chromosomal Aberrations in Wild Mice Captured in Areas Differentially Contaminated by the Fukushima Dai-Ichi Nuclear Power Plant Accident

Following the Fukushima Dai-ichi Nuclear Power Plant accident, radiation effects on nonhuman biota in the contaminated areas have been a great concern. The induction of chromosomal aberrations in splenic lymphocytes of small Japanese field mice (Apodemus argenteus) and house mice (Mus musculus) inhabiting Fukushima Prefecture was investigated. In mice inhabiting the slightly contaminated area, the average frequency of dicentric chromosomes was similar to that seen in mice inhabiting a noncontaminated control area. In contrast, mice inhabiting the moderately and heavily contaminated areas showed a significant increase in the average frequencies of dicentric chromosomes. Total absorbed dose rate was estimated to be approximately 1 mGy d(-1) and 3 mGy d(-1) in the moderately and heavily contaminated areas, respectively. Chromosomal aberrations tended to roughly increase with dose rate. Although theoretically, the frequency of chromosomal aberrations was considered proportional to the absorbed dose, chromosomal aberrations in old mice (estimated median age 300 days) did not increase with radiation dose at the same rate as that observed in young mice (estimated median age 105 days).

DNA-damage tolerance mediated by PCNA•Ub fusions in human cells is dependent on Rev1 but not Polη

In response to replication-blocking lesions, proliferating cell nuclear antigen (PCNA) can be sequentially ubiquitinated at the K164 residue, leading to two modes of DNA-damage tolerance, namely, translesion DNA synthesis (TLS) and error-free lesion bypass. Although the majority of reported data support a model whereby monoubiquitinated PCNA enhances its affinity for TLS polymerases and hence recruits them to the damage sites, this model has also been challenged by several observations. In this study, we expressed the PCNA-164R and ubiquitin (UB) fusion genes in an inducible manner in an attempt to mimic PCNA monoubiquitination in cultured human cells. It was found that expression of both N- and C-terminal PCNA•Ub fusions conferred significant tolerance to ultraviolet (UV)-induced DNA damage. Surprisingly, depletion of Polη, a TLS polymerase dedicated to bypassing UV-induced pyrimidine dimers, did not alter tolerance conferred by PCNA•Ub. In contrast, depletion of Rev1, another TLS polymerase serving as a scaffold for the assembly of the TLS complex, completely abolished PCNA•Ub-mediated damage tolerance. Similar genetic interactions were confirmed when UV-induced monoubiquitination of endogenous PCNA is abolished by RAD18 deletion. Hence, PCNA•Ub fusions bypass the requirement for PCNA monoubiquitination, and UV damage tolerance conferred by these fusions is dependent on Rev1 but independent of Polη.

Disruption of Xpg increases spontaneous mutation frequency, particularly A:T to C:G transversion.

Cells isolated from Xpg (the mouse counterpart of XPG)-disrupted mice underwent premature senescence and showed early onset of immortalization, suggesting that Xpg might be involved in genetic stability. Recent studies showed that human XPG, in addition to its function in the nucleotide excision repair (NER), was involved in the repair of oxidative base damages such as thymine glycol (Tg) and 8-oxo-guanine (8-oxoG), and this may explain the genetic instability observed in Xpg-deficient cells. To clarify this point, we determined spontaneous mutation frequencies and the type of spontaneous base substitution mutations in cells obtained from normal and Xpg-deficient mice using the supF shuttle vector (pNY200) for mutation assay. The spontaneous mutation frequency of the supF gene in pNY200 propagated in the Xpg-deficient cells was about three times higher than that in normal cells, indicating the importance of Xpg in reducing the frequency of spontaneous mutations. The frequency of spontaneous base substitution mutations at A:T sites, particularly that of the A:T to C:G transversion, increased markedly in the Xpg-deficient cells.