Yukinobu Uchiyama

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.

Two types of replication protein A in seed plants : Characterization of their functions in vitro and in vivo

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.

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.

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

Abstract DNA polymerase δ (pol δ), 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 δ from rice (Oryza sativa L. cv. Nipponbare). The open reading frames of OsPolδ1 and δ2 encoded a predicted product of 1105 amino acid residues with a molecular weight of 124 kDa for OsPolδ1, and of 429 residues with a molecular weight of 48 kDa for OsPolδ2. Northern blotting analysis indicated that OsPolδ1 and δ2 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 OsPolδ1 transcripts by in situ hybridization. In the shoot apex, OsPolδ1 mRNA was abundant in the shoot apical meristem. In the roots, the OsPolδ1 transcript accumulated at high levels in the root apical meristem. In mature leaves, OsPolδ1 was induced after UV irradiation, but OsPolδ2 was not. The amounts of the OsPolδ1 and δ2 mRNAs in the rice cells changed rapidly during cell proliferation. These results indicated that the levels of OsPolδ expression are markedly correlated with cell proliferation, and that some of OsPolδ might have special roles in the leaves.

Interaction between proliferating cell nuclear antigen (PCNA) and a DnaJ induced by DNA damage

Proliferating cell nuclear antigen (PCNA) is an essential protein for both DNA replication and DNA repair. In the present study using two-hybrid analysis with PCNA from rice, Oryza sativa L. cv. Nipponbare (OsPCNA), we found that OsPCNA interacted with rice DnaJ protein. We have identified DnaJ and designated it as OsDnaJ. OsDnaJ was able to bind to OsPCNA in vitro. Transcripts of OsDnaJ were found to be strongly expressed in the proliferating cells. mRNA of DnaJ was induced by UV and DNA-damaging agents such as H2O2. The expression patterns of OsPCNA were almost the same as OsDnaJ. The relationship between OsPCNA and OsDnaJ is discussed.

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

In plants, there are no DNA polymerase β (Pol β) 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 β 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, H2O2 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.

β-Sitosterol-3-O-β-D-glucopyranoside : A eukaryotic DNA polymerase λ 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.

Dielectric Relaxation Analysis of Single-Stranded DNA in Liquid Crystals

Since liquid crystals have found wide use in liquid crystal displays, we try to apply liquid crystals to biotechnology. The purpose of this study is to describe the physical properties of the biopolymer DNA in regard to electrooptics. Synthetic oligonucleotide molecules were dispersed in a nematic liquid crystal (NLC). The ICP analyses showed that all of the oligonuleotide molecules added dispersed in the NLC. This sample was injected into the twisted nematic liquid crystal display cells, and then we measured its dielectric properties. The NLC doped with oligonucleotide molecules exhibited dielectric relaxation characteristics, depending on the concentration of oligonucleotide molecules. After analyzing this effect by using the equivalent circuit model, we found a difference in electrical conductivity between Watson–Crick base pairs (AT and GC). This result suggests that the difference might have contributed to the existence of a functional group involved in the hydrogen-bond interaction.

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

While mammalian DNA polymerase β (Pol β), 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 β from jellyfish Aurelia sp.1. (AsPol β). Despite of phylogenetic distance and environmental differences between jellyfish and mammals, in vitro assays showed biochemical characteristics of AsPol β 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 β and the other members of the Pol X family.