Electronic Structure of Ce-Doped and -Undoped Nd_{2}CuO_{4} Superconducting Thin Films Studied by Hard X-Ray Photoemission and Soft X-Ray Absorption Spectroscopy.

In order to realize superconductivity in cuprates with the T^{'}-type structure, not only chemical substitution (Ce doping) but also postgrowth reduction annealing is necessary. In the case of thin films, however, well-designed reduction annealing alone without Ce doping can induce superconductivity in the T^{'}-type cuprates. In order to unveil the origin of superconductivity in the Ce-undoped T^{'}-type cuprates, we have performed bulk-sensitive hard x-ray photoemission and soft x-ray absorption spectroscopy on superconducting and nonsuperconducting Nd_{2-x}Ce_{x}CuO_{4} (x=0, 0.15, and 0.19) thin films. By postgrowth annealing, core-level spectra exhibited dramatic changes, which we attributed to the enhancement of core-hole screening in the CuO_{2} plane and the shift of chemical potential along with changes in the band filling. The result suggests that the superconducting Nd_{2}CuO_{4} film is doped with electrons despite the absence of the Ce substitution.

Direct k-space mapping of the electronic structure in an oxide-oxide interface.

The interface between LaAlO(3) and SrTiO(3) hosts a two-dimensional electron system of itinerant carriers, although both oxides are band insulators. Interface ferromagnetism coexisting with superconductivity has been found and attributed to local moments. Experimentally, it has been established that Ti 3d electrons are confined to the interface. Using soft x-ray angle-resolved resonant photoelectron spectroscopy we have directly mapped the interface states in k space. Our data demonstrate a charge dichotomy. A mobile fraction contributes to Fermi surface sheets, whereas a localized portion at higher binding energies is tentatively attributed to electrons trapped by O vacancies in the SrTiO(3). While photovoltage effects in the polar LaAlO(3) layers cannot be excluded, the apparent absence of surface-related Fermi surface sheets could also be fully reconciled in a recently proposed electronic reconstruction picture where the built-in potential in the LaAlO(3) is compensated by surface O vacancies serving also as a charge reservoir.

Photic induction of mPer1 and mPer2 in cry-deficient mice lacking a biological clock.

Mice lacking mCry1 and mCry2 are behaviorally arrhythmic. As shown here, cyclic expression of the clock genes mPer1 and mPer2 (mammalian Period genes 1 and 2) in the suprachiasmatic nucleus and peripheral tissues is abolished and mPer1 and mPer2 mRNA levels are constitutively high. These findings indicate that the biological clock is eliminated in the absence of both mCRY1 and mCRY2 (mammalian cryptochromes 1 and 2) and support the idea that mammalian CRY proteins act in the negative limb of the circadian feedback loop. The mCry double-mutant mice retain the ability to have mPer1 and mPer2 expression induced by a brief light stimulus known to phase-shift the biological clock in wild-type animals. Thus, mCRY1 and mCRY2 are dispensable for light-induced phase shifting of the biological clock.

DNA Polymerase Beta Participates in Mitochondrial DNA Repair.

We have detected DNA polymerase beta (Polß), known as a key nuclear base excision repair (BER) protein, in mitochondrial protein extracts derived from mammalian tissue and cells. Manipulation of the N-terminal sequence affected the amount of Polß in the mitochondria. Using Polß fragments, mitochondrion-specific protein partners were identified, with the interactors functioning mainly in DNA maintenance and mitochondrial import. Of particular interest was the identification of the proteins TWINKLE, SSBP1, and TFAM, all of which are mitochondrion-specific DNA effectors and are known to function in the nucleoid. Polß directly interacted functionally with the mitochondrial helicase TWINKLE. Human kidney cells with Polß knockout (KO) had higher endogenous mitochondrial DNA (mtDNA) damage. Mitochondrial extracts derived from heterozygous Polß mouse tissue and KO cells had lower nucleotide incorporation activity. Mouse-derived Polß null fibroblasts had severely affected metabolic parameters. Indeed, gene knockout of Polß caused mitochondrial dysfunction, including reduced membrane potential and mitochondrial content. We show that Polß is a mitochondrial polymerase involved in mtDNA maintenance and is required for mitochondrial homeostasis.

Repair of apurinic/apyrimidinic sites by UV damage endonuclease; a repair protein for UV and oxidative damage.

UV damage endonuclease (UVDE) initiates a novel form of excision repair by introducing a nick imme-diately 5" to UV-induced cyclobutane pyrimidine dimers or 6-4 photoproducts. Here, we report that apurinic/apyrimidinic (AP) sites are also nicked by Neurospora crassa and Schizosaccharomyces pombe UVDE. UVDE introduces a nick immediately 5" to the AP site leaving a 3"-OH and a 5"-phosphate AP. Apyrimidinic sites are more effectively nicked by UVDE than apurinic sites. UVDE also possesses 3"-repair activities for AP sites nicked by AP lyase and for 3"-phosphoglycolate produced by bleomycin. The Uvde gene introduced into Escherichia coli cells lacking two types of AP endonuclease, Exo III and Endo IV, gave the host cells resistance to methylmethane sulfonate and t-butyl hydroperoxide. We identified two AP endonuclease activities in S.pombe cell extracts. Besides cyclobutane pyrimidine dimers and 6-4 photoproducts, N. crassa UVDE also nicks Dewar photoproducts. Thus, UVDE is able to repair both of the major forms of DNA damage in living organisms: UV-induced DNA lesions and AP sites.

Differential subcellular localization of human MutY homolog (hMYH) and the functional activity of adenine:8-oxoguanine DNA glycosylase.

The post-replicative adenine:8-oxo-7,8-dihydroguanine (GO) mismatch is crucial for G:C to T:A transversion. This mismatch is corrected by Escherichia coli MutY which excises the adenine from A:GO. A candidate gene coding for the human counterpart of MutY has been cloned as hMYH. However, the function and enzyme activities of the gene product have not been identified. We previously demonstrated that an epitope-tagged hMYH protein behaves as a mitochondrial protein. In the present study, we have identified an alternative hMYH transcript, termed type 2, which differs in the exon 1 sequence of the known transcript (type 1). A nuclear localization for the type 2 protein was revealed by detection of epitope-tagged protein in COS-7 cells. Expression of both type 1 and type 2 transcripts was reduced in post-mitotic tissues. hMYH cDNA suppressed the mutator phenotype of E.coli mutY. In vitro expressed hMYH showed adenine DNA glycosylase activity toward the A:GO substrate. The protein can bind to A:GO, and to T:GO and G:GO without apparent catalysis. These results represent the first demonstration of the function of the hMYH gene product which is differentially transported into the nucleus or the mitochondria by alternative splicing

Escherichia coli Nth and human hNTH1 DNA glycosylases are involved in removal of 8-oxoguanine from 8-oxoguanine/guanine mispairs in DNA.

The spectrum of DNA damage caused by reactive oxygen species includes a wide variety of modifications of purine and pyrimidine bases. Among these modified bases, 7,8-dihydro-8-oxoguanine (8-oxoG) is an important mutagenic lesion. Base excision repair is a critical mechanism for preventing mutations by removing the oxidative lesion from the DNA. That the spontaneous mutation frequency of the Escherichia coli mutT mutant is much higher than that of the mutM or mutY mutant indicates a significant potential for mutation due to 8-oxoG incorporation opposite A and G during DNA replication. In fact, the removal of A and G in such a situation by MutY protein would fix rather than prevent mutation. This suggests the need for differential removal of 8-oxoG when incorporated into DNA, versus being generated in situ. In this study we demonstrate that E.coli Nth protein (endonuclease III) has an 8-oxoG DNA glycosylase/AP lyase activity which removes 8-oxoG preferentially from 8-oxoG/G mispairs. The MutM and Nei proteins are also capable of removing 8-oxoG from mispairs. The frequency of spontaneous G:C-->C:G transversions was significantly increased in E.coli CC103mutMnthnei mutants compared with wild-type, mutM, nth, nei, mutMnei, mutMnth and nthnei strains. From these results it is concluded that Nth protein, together with the MutM and Nei proteins, is involved in the repair of 8-oxoG when it is incorporated opposite G. Furthermore, we found that human hNTH1 protein, a homolog of E.coli Nth protein, has similar DNA glycosylase/AP lyase activity that removes 8-oxoG from 8-oxoG/G mispairs.

Highly elevated ultraviolet-induced mutation frequency in isolated Chinese hamster cell lines defective in nucleotide excision repair and mismatch repair proteins.

We have isolated N-methyl-N'-nitro-N-nitrosoguanidine-resistant cell lines from 43-3B Chinese hamster ovary cells, which are deficient in the ERCC1 gene involved in nucleotide excision repair. By Western blotting analysis, we found cell lines that are deficient or decreased in the amount of MSH6, or PMS2, or MSH2 proteins. Cell extracts of these cell lines show reduced efficiency of G:T mismatch repair activity. Compared with 43-3B, these cell lines exhibit highly elevated UV-induced mutation rates, indicating that mammalian mismatch repair can suppress UV-induced mutagenesis and may play a role in the fidelity of DNA replication at the sites of UV damage.

Identification of the eleventh complementation group of UV-sensitive excision repair-defective rodent mutants.

The drug-sensitive mutant UVS1, isolated from the Chinese hamster cell line CHO9, was previously found to complement the UV sensitivity of the excision repair-defective rodent mutants representative of groups 1 to 8 (Hata et al., Cancer Res., 51: 195-198, 1991; M. Numata et al., personal communication). Recently two new complementation groups of UV-sensitive CHO mutants, e.g., groups 9 and 10, have been identified (Stefanini et al., Cancer Res., 51: 3965-3971, 1991). In this paper we demonstrate that the repair defect in UVS1 cells is genetically different from those present in the mutants CHO7PV and CHO4PV, representing groups 9 and 10, respectively. Therefore, UVS1 represents a new complementation group of UV-sensitive rodent cell lines, the eleventh group.

Isolation of two chloroethylnitrosourea-sensitive Chinese hamster cell lines.

1-[(4-Amino-2-methylpyrimidin-5-yl)methyl]-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU), a cancer chemotherapeutic bifunctional alkylating agent, causes chloroethylation of DNA and subsequent DNA strand cross-linking through an ethylene bridge. We isolated and characterized two ACNU-sensitive mutants from mutagenized Chinese hamster ovary cells and found them to be new drug-sensitive recessive Chinese hamster mutants. Both mutants were sensitive to various monofunctional alkylating agents in a way similar to that of the parental cell lines CHO9. One mutant (UVS1) was cross-sensitive to UV and complemented the UV sensitivity of all Chinese hamster cell lines of 7 established complementation groups. Since UV-induced unscheduled DNA synthesis was very low, a new locus related to excision repair is thought to be defective in this cell line. Another ACNU-sensitive mutant, CNU1, was slightly more sensitive to UV than the parent cell line. CNU1 was cross-sensitive to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and slightly more sensitive to mitomycin C. No increased accumulation of ACNU and a low level of UV-induced unscheduled DNA synthesis in this cell as compared with the parental cell line suggest that there is abnormality in a repair response of this mutant cell to some types of DNA cross-links.

Identification of cellular defect in UVS1, a UV-sensitive Chinese hamster ovary mutant cell line.

UVS1 is an intermediately UV-sensitive Chinese hamster ovary mutant originally isolated by its hypersensitivity to an anticancer drug, 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosour ea hydrochloride. By cell fusion analysis, UVS1 complemented the UV sensitivity of the mouse lymphoma cell line US31 from the eighth complementation group of UV-sensitive rodent cell lines. By enzyme-linked immunosorbent assay we found that within 3 h after UV irradiation both pyrimidine dimers and (6-4)photoproducts in UVS1 were not removed from chromosomal DNA in UVS1 at all. Twenty-four h after UV irradiation the removal rate of (6-4)photoproducts was intermediate between CHO9, the parental cell line, and 43-3B, a UV-hypersensitive Chinese hamster ovary mutant of the complementation group 1, whereas the pyrimidine dimers in UVS1 were removed less efficiently as 43-3B. Alkaline elution assay showed that the incising activity to damaged DNA after UV irradiation of UVS1 was as low as that of 43-3B. The number of 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosour ea hydrochloride-induced DNA interstrand cross-links of UVS1 was almost equal to that of 43-3B and about 1.5 times more than that of CHO9, suggesting that the gene products defective in UVS1 and 43-3B are essential for the excision repair of DNA damages produced by 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosour ea hydrochloride.

Cloning and characterization of mammalian 8-hydroxyguanine-specific DNA glycosylase/apurinic, apyrimidinic lyase, a functional mutM homologue.

8-Hydroxyguanine (8-OH-G) is one of the major DNA oxidation products implicated in mutagenesis induced by oxygen radical-forming agents, including ionizing radiation. It is also believed to be involved in spontaneous mutation induced by metabolically produced oxygen radicals. A mammalian homologue of 8-OH-G glycosylase/apurinic, apyrimidinic lyase (mutM homologue, MMH) has been identified in the EST database (for expressed sequence tags) through a homology search with yeast OGG1 protein. The human MMH protein (hMMH), 34% identical to the yeast OGG1 protein, is a member of the DNA repair protein superfamily. The hMMH gene was composed of seven exons, with the alternate last exon, exon 8, producing three major alternative splicing isoforms, because splicing of the sixth intron was optional. The hMMH protein expressed in Escherichia coli revealed the glycosylase activity and apurinic, apyrimidinic lyase activity on duplex DNA containing 8-OH-G. The hMMH protein can rescue a spontaneous mutator strain of E. coli lacking mutM and mutY. By the introduction of recombinant hMMH, the rate of mutation, the formation of rifampicin-resistant revertants, was reduced by 4-7 fold. Genomic structure analysis showed that 3' exons of the hMMH gene are transcribed on the antisense strand of the calcium-dependent calmodulin kinase 1 gene.

Photorepair prevents ultraviolet-induced apoptosis in human cells expressing the marsupial photolyase gene.

Photolyase absorbs blue light and employs the energy to remove UV-induced DNA damage, cyclobutane pyrimidine dimers, or pyrimidine pyrimidone (6-4) lesions. These enzymes have been found in many living organisms ranging from bacteria to aplacental mammals, but their photoreactivation effect, such as survival increase of UV-irradiated cells by light-illumination, has not been identified in placental mammals, including humans. Therefore, we introduced a photolyase gene derived from the marsupial rat kangaroo, Potorous tridactylus, into HeLa cells and established the first human cell line capable of photorepairing UV-induced pyrimidine dimers. Several clones were found to increase cell survival after UV irradiation when illuminated by fluorescent light. The induction of apoptosis by UV irradiation was investigated in these photoreactivation-proficient cells. Several typical features of the programmed cell death, such as internucleosomal DNA degradation, presence of subdiploid cells, loss of membrane integrity, and chromosomal condensation, were found to be induced by UV in the HeLa cells, but they can be reduced by photorepair. This implicates that cyclobutane pyrimidine dimers cause UV-induced apoptosis in human cells.

Photorepair of RNA polymerase arrest and apoptosis after ultraviolet irradiation in normal and XPB deficient rodent cells.

Cyclobutane pyrimidine dimers (CPDs) are directly involved in signaling for UV-induced apoptosis in mammalian cells. Failure to remove these lesions, specially those located at actively expressing genes, is critical, as cells defective in transcription coupled repair have increased apoptotic levels. Thus, the blockage of RNA synthesis by lesions is an important candidate event triggering off active cell death. In this work, wild-type and XPB mutated Chinese hamster ovary (CHO) cells expressing a marsupial photolyase, that removes specifically CPDs from the damaged DNA, were generated, in order to investigate the importance of this lesion in both RNA transcription blockage and apoptotic induction. Photorepair strongly recovers RNA synthesis in wild-type CHO cell line, although the resumption of transcription is decreased in XPB deficient cells. This recovery is accompanied by the prevention of cells entering into apoptosis. These results demonstrate that marsupial photolyase has access to CPDs blocking RNA synthesis in vivo, and this may be affected by the presence of a mutated XPB protein.

The DNA damage spectrum produced by simulated sunlight.

The mutagenic effects of ultraviolet and solar irradiation are thought to be due to the formation of DNA photoproducts, most notably cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)PPs). Experimental systems for determining the levels and sequence dependence of photoproduct formation in DNA have often used high doses of short-wave (UVC) irradiation. We have re-assessed this issue by using DNA sequencing technologies and different doses of UVC as well as more physiologically relevant doses of solar irradiation emitted from a solar UV simulator. It has been questioned whether hot alkali treatment can detect (6-4)PPs at all sequence positions. With high UVC doses, the sequence distribution of (6-4)PPs was virtually identical when hot alkali or UV damage endonuclease (UVDE) were used for detection, which appears to validate both methods. The (6-4)PPs form at 5'-TpC and 5'CpC sequences but very low levels are seen at all other dipyrimidines including 5'-TpT. Contrary to expectation, we find that (6-4) photoproducts form at almost undetectable levels under conditions of irradiation for up to five hours with the solar UV simulator. The same treatment produces high levels of CPDs. In addition, DNA glycosylases, which recognize oxidized and ring-opened bases, did not produce significant cleavage of sunlight-irradiated DNA. From these data, we conclude that cyclobutane pyrimidine dimers are at least 20 to 40 times more frequent than any other DNA photoproduct when DNA or cells are irradiated with simulated sunlight.

Crystallization and preliminary X-ray diffraction studies of photolyase (photoreactivating enzyme) from the cyanobacterium Anacystis nidulans.

Photolyase (photoreactivating enzyme) from the cyanobacterium Anacystis nidulans was crystallized by the hanging drop vapor diffusion procedure using ammonium sulfate as a precipitant. The pale-yellow crystals were grown to a size of 0.4 mm in length and 0.1 mm in diameter. They belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 90.7 A and c = 135 A. Assuming that the asymmetric unit contains one molecule, the Vm value is calculated as 2.6 A3/dalton. The crystals are stable towards X-ray exposure and diffract beyond 2.5 A resolution.

Cloning and characterization of a mouse homologue (mNthl1) of Escherichia coli endonuclease III.

Endonuclease III (endoIII; nth gene product) of Escherichia coli is known to be a DNA repair enzyme having a relatively broad specificity for damaged pyrimidine bases of DNA. Here, we describe the cloning and characterization of the cDNA and the gene for a mouse homologue (mNthl1/mNth1) of endoIII. The cDNA was cloned from a mouse T-cell cDNA library with a probe prepared by PCR using the library and specific PCR primers synthesized based on the reported information of partial amino acid sequences of bovine NTHL1/NTH1 and of EST Data Bases. The cDNA is 1025 nucleotides long and encodes a protein consisting of 300 amino acids with a predicted molecular mass of 33.6 kDa. The amino acid sequence exhibits significant homologies to those of endoIII and its prokaryotic and eukaryotic homologues. The recombinant mNthl1 with a hexahistidine tag was overexpressed in a nth::cmr nei::Kmr double mutant of E. coli, and purified to apparent homogeneity. The enzyme showed thymine glycol DNA glycosylase, urea DNA glycosylase and AP lyase activities. Northern blot analysis indicated that mNthl1 mRNA is about 1 kb and is expressed ubiquitously. A 15 kb DNA fragment containing the mNthl1 gene was cloned from a mouse genomic library and sequenced. The gene consists of six exons and five introns spanning 6.09 kb. The sequenced 5' flanking region lacks a typical TATA box, but contains a CAAT box and putative binding sites for several transcription factors such as Ets, Sp1, AP-1 and AP-2. The mNthl1 gene was shown to lie immediately adjacent to the tuberous sclerosis 2 (Tsc2) gene in a 5'-to-5' orientation by sequence analysis and was assigned to chromosome 17A3 by in situ hybridization.

Homology between the photoreactivation genes of Saccharomyces cerevisiae and Escherichia coli.

A cloned fragment of Saccharomyces cerevisiae chromosomal DNA carrying the photoreactivation gene (PHR) has been sequenced. The fragment contains a 1695-bp intronless open reading frame (ORF) coding for a polypeptide of 564 amino acids (aa). The phr gene of Escherichia coli was also sequenced, and the sequence is in agreement with the published data. The yeast PHR gene has a G + C content of 36.2%, whereas 53.7% was found for the E. coli gene. Despite the difference in G + C content there is a 35% homology between the deduced aa sequences. This homology suggests that both genes have originated from a common ancestral gene.

A gene family homologous to the S-phase specific gene in higher plants is essential for cell proliferation in Saccharomyces cerevisiae.

Previously we reported the isolation and characterization of the gene, cyc07, which was specifically expressed in the S phase during the cell cycle in synchronous cell division cultures of the higher plant, Catharanthus roseus. We found that the yeast Saccharomyces cerevisiae contains two closely related genes which show a high degree of similarity (about 64% at the amino acid level) to cyc07 of C. roseus. Site-directed disruption mutations demonstrated that the two yeast genes, homologous to cyc07, constitute an essential gene family for cell proliferation in yeast cells. Furthermore, the rate of cell proliferation varied with the gene copy number.