Characterization of marine X-family DNA polymerases and comparative analysis of base excision repair proteins.

While mammalian DNA polymerase ß (Pol ß), which is a member of the Pol X family, play important roles in base excision repair (BER) that efficiently removes DNA base lesions arising from both endogenous and exogenous agents, this protein has been found only a subset of animals. To understand natural evolution of this enzyme, we isolated and characterized Pol ß from jellyfish Aurelia sp.1. (AsPol ß). Despite of phylogenetic distance and environmental differences between jellyfish and mammals, in vitro assays showed biochemical characteristics of AsPol ß were very similar to those of a mammalian counterpart. We also searched two other homologs of mammalian genes that were involved in short patch (sp) BER in the nucleotide sequence database, and found that both of these homologs were encoded in the genomes of a lineage from Cnidarians through mammals and Arthropods. This study suggests that a DNA repair mechanism resembling mammalian sp-BER may be largely limited to a subset of animals. On the basis of our findings and previous reports, we discuss possible evolutional model of Pol ß and the other members of the Pol X family.

Higher plant RecA-like protein is homologous to RadA.

A novel RecA-like protein, differing from Dmc1 and Rad51, was characterized in Oryza sativa L. cv. Nipponbare. Because the protein is homologous to bacterial RadA, the gene was designated OsRadA. The open reading frame was predicted to encode a 66kDa protein of 619 amino acid residues and was found in plants but not animals or yeast. OsRadA showed D-loop and single-stranded DNA-dependent ATPase activities. Gene expression was found to be high in meristematic tissues, and was localized in the nucleus. An RNAi mutant of Arabidopsis thaliana RadA (AtRadA) was sensitive to mutagenic agents such as UV and MMC, suggesting that RadA functions in DNA repair.

Selective inhibitors of terminal deoxyribonucleotidyltransferase (TdT): baicalin and genistin.

Studies of mammalian terminal deoxyribonucleotidyltransferase (TdT) are facilitated by use of inhibitors that selectively knock down the activity of the enzyme. We have screened for selective inhibitors of TdT and identified a natural compound with this property in the Japanese vegetable, Arctium lappa. The compound has little effect on the activities of mammalian DNA polymerases, such as alpha, beta, delta or lambda polymerase, and prokaryotic DNA polymerases, such as Taq DNA polymerase, T4 DNA polymerase and Klenow fragment. H1- and C13-NMR spectroscopic analyses showed the compound to be baicalin, a compound previously reported as an anti-inflammatory or antipyretic agent. The IC50 value of baicalin to TdT was 18.6 microM. We also found that genistin, a baicalin derivative known to be antimutagenic, more selectively inhibited TdT activity than baicalin, although its IC50 value was weaker (28.7 microM). Genistin and baicalin also inhibited the activity of truncated TdT (the so-called pol beta core domain) in which the BRCT motif was deleted in its N-terminal region. In kinetic analyses, inhibition by either genistin or baicalin was competitive with the primer and non-competitive with the dNTP substrate. The compounds may, therefore, bind directly to the primer-binding site of TdT and simultaneously disturb dNTP substrate incorporation into the primer. Genistin and baicalin should prove to be useful agents for studying TdT.

Beta-sitosterol-3-O-beta-D-glucopyranoside: a eukaryotic DNA polymerase lambda inhibitor.

Beta-sitosterol-3-O-beta-D-glucopyranoside (compound 1), a steroidal glycoside isolated from onion (Allium cepa L.) selectively inhibited the activity of mammalian DNA polymerase lambda (pol lambda) in vitro. The compound did not influence the activities of replicative DNA polymerases such as alpha, delta and epsilon, but also showed no effect even on the activity of pol beta which is thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. Since parts of compound 1 such as beta-sitosterol (compound 2) and D-glucose (compound 3) did not influence the activities of any enzymes tested, the converted structure of compounds 2 and 3 might be important for pol lambda inhibition. The inhibitory effect of compound 1 on both intact pol lambda (i.e. residues 1-575) and a truncated pol lambda lacking the N-terminal BRCA1 C-terminus (BRCT) domain (133-575, del-1 pol lambda) was dose-dependent, and 50% inhibition was observed at a concentration of 9.1 and 5.4 microM, respectively. The compound 1-induced inhibition of del-1 pol lambda activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol lambda inhibitory mechanism of compound 1 is discussed.

A novel DNA polymerase homologous to Escherichia coli DNA polymerase I from a higher plant, rice (Oryza sativa L.).

A novel DNA polymerase, designated as OsPolI-like, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare). The OsPolI-like cDNA was 3765 bp in length, and the open reading frame encoded a predicted product of 977 amino acid residues with a molecular weight of 100 kDa. The OsPolI-like gene has been mapped to chromosome 8 and contains 12 exons and 11 introns. The encoded protein showed a high degree of sequence and structural homology to Escherichia coli pol I protein, but differed from DNA polymerase gamma and theta. The DNA polymerase domain of OsPolI-like showed DNA polymerase activity. Subcellular fractionation analysis suggested that the protein is localized in the plastid. Northern and western blotting, and in situ hybridization analyses demonstrated preferential expression of OsPolI-like in meristematic tissues such as shoot apical meristem, root apical meristem, leaf primordia and the marginal meristem. Interestingly, no expression was detected in mature leaves, although they have a high chloroplast content. These properties indicated that OsPolI-like is a novel plant DNA polymerase. The function of OsPolI-like is discussed in relation to plastid maturation.

[Nosocomial infection by multidrug-resistant Pseudomonas Aeruginosa (MDRP) presumably spread a radiation technician].

Isolated of multidrug resistance Pseudomonas aeruginosa (MDRP) that the receptivity pattern of the antimicrobial suscepti respectively resembled isolated from clinical specimens (sputum) in two patients of each internal medicine ward in Kitasato University East Hospital for two days from September 18 and 20, 2004. Both of bacteria were formed small colonies of a smooth-type on dollargalluskey improvement-type BTB agar plates, and the judgment of ClassB (metallo)-beta-lactamase by biochemical properties and disk diffusion method sodium mercaoto-acetic acid (SMA) was mutually corresponding. Moreover, it was same serotype C according to the serotype, and it was confirmed that it was the same bacterial strain from the molecular epidemiology analysis by Random amplified polymorphic DNA polymerase chain reaction (Random amplified polymorphic DNA polymerase chain reaction: RAPD). From the investigation of clinical backgrounds of two patients who isolated bacterial strains, September 18, 2004. 10 : 20 a.m., and 10 : 40 a.m., other chances that can become with contact infection in this hospital, except conducted X-Ray or roentgenograph of the chest and abdomen of Portable X-ray device continuously done by one radiation technician was not seen. Because it had turned out that a radiation technician who had taken charge had been neglecting the hand washing at the time of each X-Ray or roentgenograph, it was guessed the case with nosocomial infection by contact infection occurred via specific radiation technician.

Two types of replication protein A in seed plants.

Replication protein A (RPA), a heterotrimeric protein composed of 70, 32 and 14-kDa subunits, has been shown to be essential for DNA replication, repair, recombination, and transcription. Previously, we found that, in two seed plants, rice and Arabidopsis, there are two different types of RPA70-kDa subunit. Substantial biochemical and genetic characterization of these two subunits, termed OsRPA70a and OsRPA70b or AtRPA70a and AtRPA70b, respectively, is described in this report. Inactivation of AtRPA70a by transfer DNA insertion or RNA interference is lethal, so the complex containing RPA70a may be essential for DNA replication. Transfer DNA insertion and RNAi lines for AtRPA70b are morphologically normal, albeit hypersensitive to certain mutagens, such as UV-B and methyl methanesulfonate, suggesting that RPA70b functions mostly in DNA repair. In two-hybrid, pull-down and coexpression analysis, OsRPA70b was found to interact more selectively than OsRPA70a with OsRPA32. The data suggest that two different types of RPA heterotrimer are present in seed plants, and that there may be additional 32 and 14-kDa subunit homologs that interact with OsRPA70a. Each of the two probable plant RPA complexes may have different roles in DNA metabolism.

Characterization of DNA polymerase delta from a higher plant, rice (Oryza sativa L.).

DNA polymerase delta (pol delta), which is comprised of at least two essential subunits, is an important enzyme involved in DNA replication and repair. We have cloned and characterized both the catalytic and small subunits of pol delta from rice (Oryza sativa L. cv. Nipponbare). The open reading frames of OsPoldelta1 and delta2 encoded a predicted product of 1105 amino acid residues with a molecular weight of 124 kDa for OsPoldelta1, and of 429 residues with a molecular weight of 48 kDa for OsPoldelta2. Northern blotting analysis indicated that OsPoldelta1 and delta2 transcripts were expressed strongly in proliferating tissues such as shoot apical meristem. The expression patterns of both subunits in the organs were slightly different. Therefore, we analyzed the spatial distribution pattern of OsPoldelta1 transcripts by in situ hybridization. In the shoot apex, OsPoldelta1 mRNA was abundant in the shoot apical meristem. In the roots, the OsPoldelta1 transcript accumulated at high levels in the root apical meristem. In mature leaves, OsPoldelta1 was induced after UV irradiation, but OsPoldelta2 was not. The amounts of the OsPoldelta1 and delta2 mRNAs in the rice cells changed rapidly during cell proliferation. These results indicated that the levels of OsPoldelta expression are markedly correlated with cell proliferation, and that some of OsPoldelta might have special roles in the leaves.

Rice UV-damaged DNA binding protein homologues are most abundant in proliferating tissues.

Ultraviolet-damaged DNA binding protein (UV-DDB) is an important factor involved in DNA repair. To study the role of UV-DDB, we attempted to obtain the cDNA and the protein of a plant UV-DDB. We succeeded in isolating both genes for UV-DDB subunits from rice (Oryza sativa cv. Nipponbare), designated as OsUV-DDB1 and OsUV-DDB2. OsUV-DDB2 (65 kDa) was much larger than human UV-DDB2, but immunoprecipitation and gel mobility shift assay suggested that OsUV-DDB2 is a plant counterpart of UV-DDB2. The transcripts were expressed in proliferating tissues such as the meristem, but were detected at only low levels in the mature leaves, although the leaves are strongly exposed to UV. These transcripts were induced in the meristem after UV-irradiation. The expression levels of OsUV-DDB were significantly reduced when cell proliferation was temporarily halted. These results indicated that the level of OsUV-DDB expression is correlated with cell proliferation, and its expression may be required mostly for DNA repair in DNA replication.

The multi-replication protein A (RPA) system--a new perspective.

Replication protein A (RPA) complex has been shown, using both in vivo and in vitro approaches, to be required for most aspects of eukaryotic DNA metabolism: replication, repair, telomere maintenance and homologous recombination. Here, we review recent data concerning the function and biological importance of the multi-RPA complex. There are distinct complexes of RPA found in the biological kingdoms, although for a long time only one type of RPA complex was believed to be present in eukaryotes. Each complex probably serves a different role. In higher plants, three distinct large and medium subunits are present, but only one species of the smallest subunit. Each of these protein subunits forms stable complexes with their respective partners. They are paralogs as complex. Humans possess two paralogs and one analog of RPA. The multi-RPA system can be regarded as universal in eukaryotes. Among eukaryotic kingdoms, paralogs, orthologs, analogs and heterologs of many DNA synthesis-related factors, including RPA, are ubiquitous. Convergent evolution seems to be ubiquitous in these processes. Using recent findings, we review the composition and biological functions of RPA complexes.

Distribution and roles of X-family DNA polymerases in eukaryotes.

Four types of DNA polymerase (Pol beta, Pol lambda, Pol mu and TdT) have been identified in eukaryotes as members of the polymerase X-family. Only vertebrates have all four types of enzyme. Plants and fungi have one or two X-family polymerases, while protostomes, such as fruit flies and nematodes, do not appear to have any. It is possible that the well-known metabolic pathways in which these enzymes are involved are restricted to the vertebrate world. The distribution of the DNA polymerases involved in DNA repair across the various biological kingdoms differs from that of the DNA polymerases involved in chromosomal DNA replication. In this review, we focus on the interesting pattern of distribution of the X-family enzymes across biological kingdoms and speculate on their roles.

Characterization of plant XRCC1 and its interaction with proliferating cell nuclear antigen.

In plants, there are no DNA polymerase beta (Pol beta) and DNA ligase III (Lig3) genes. Thus, the plant short-patch base excision repair (short-patch BER) pathway must differ considerably from that in mammals. We characterized the rice (Oryza Sativa L. cv. Nipponbare) homologue of the mammalian X-ray repair cross complementing 1 (XRCC1), a well-known BER protein. The plant XRCC1 lacks the N-terminal domain (NTD) which is required for Pol beta binding and is essential for mammalian cell survival. The recombinant rice XRCC1 (OsXRCC1) protein binds single-stranded DNA (ssDNA) as well as double-stranded DNA (dsDNA) and also interacts with rice proliferating cell nuclear antigen (OsPCNA) in a pull-down assay. Through immunoprecipitation, we demonstrated that OsXRCC1 forms a complex with PCNA in vivo. OsXRCC1 mRNA was expressed in all rice organs and was induced by application of bleomycin, but not of MMS, H(2)O(2) or UV-B. Bleomycin also increased the fraction of OsXRCC1 associated with chromatin. These results suggest that OsXRCC1 contributes to DNA repair pathways that differ from the mammalian BER system.

Characterization of T-DNA insertion mutants and RNAi silenced plants of Arabidopsis thaliana UV-damaged DNA binding protein 2 (AtUV-DDB2).

The human UV-damaged DNA binding protein (UV-DDB), a heterodimeric protein composed of 127 kDa (UV-DDB1) and 48 kDa (UV-DDB2) subunits, has been shown to be involved in DNA repair. To elucidate the in vivo function of plant UV-DDB2, we have analyzed T-DNA insertion mutants of the Arabidopsis thaliana UV-DDB2 subunit (atuv-ddb2 mutants) and AtUV-DDB2 RNAi silenced plants (atuv-ddb2 silenced plants). atuv-ddb2 mutants and atuv-ddb2 silenced plants were both viable, suggesting that AtUV-DDB2 is not essential for survival. Interestingly, both plant types showed a dwarf phenotype, implying impaired growth of the meristem. To the best of our knowledge, this is the first occasion that a dwarf phenotype has been found to be associated with a UV-DDB2 mutation in either plants or animals. The mutants also demonstrated increased sensitivity to UV irradiation, methyl methanesulfonate and hydrogen peroxide treatment, indicating that AtUV-DDB2 is also involved in DNA repair. Our results lead us to suggest that not only does AtUV-DDB2 function in DNA repair, it also has a direct or indirect influence on cell proliferation in the plant meristem.

Characterization of four RecQ homologues from rice (Oryza sativa L. cv. Nipponbare).

The RecQ family of DNA helicases is conserved throughout the biological kingdoms. In this report, we have characterized four RecQ homologues clearly expressed in rice. OsRecQ1, OsRecQ886, and OsRecQsim expressions were strongly detected in meristematic tissues. Transcription of the OsRecQ homologues was differentially induced by several types of DNA-damaging agents. The expression of four OsRecQ homologues was induced by MMS and bleomycin. OsRecQ1 and OsRecQ886 were induced by H(2)O(2), and MitomycinC strongly induced the expression of OsRecQ1. Transient expression of OsRecQ/GFP fusion proteins demonstrated that OsRecQ2 and OsRecQ886 are found in nuclei, whereas OsRecQ1 and OsRecQsim are found in plastids. Neither OsRecQ1 nor OsRecQsim are induced by light. These results indicate that four of the RecQ homologues have different and specific functions in DNA repair pathways, and that OsRecQ1 and OsRecQsim may not involve in plastid differentiation but different aspects of a plastid-specific DNA repair system.