Tomoyuki Furukawa

Two types of replication protein A 70 kDa subunit in rice, Oryza sativa: molecular cloning, characterization, and cellular & tissue distribution.

Replication protein A (RPA), which is comprised of three subunits, is an important factor involved in DNA replication, repair, and transcription. We isolated and characterized 70 and 32 kDa subunits of RPA from rice (Oryza sativa cv. Nipponbare) termed OsRPA70a and OsRPA32. OsRPA70a shows a low level of homology with OsRPA1 which was isolated from deepwater rice (Oryza sativa cv. Pin Gaew 56), previously. We also succeeded to isolate OsRPA70b which is homologue to OsRPA1 from Oryza sativa cv. Nipponbare. OsRPA70a shows only 33.8% sequence identity with OsRPA70b, indicating that two different types of 70 kDa RPA subunits are present in Oryza sativa cv. Nipponbare. These subunits showed differences in their expression patterns among tissues. The transcripts of OsRPA70a and OsRPA32 were expressed strongly in proliferating tissues such as root tips and young leaves that contain root apical meristem and marginal meristem, respectively, and weakly in the mature leaves which have no proliferating tissues. On the other hand, OsRPA70b was expressed mostly in the proliferating tissues. The roles of these molecules in plant DNA replication and DNA repair are discussed.

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.

Functional characterization of two flap endonuclease-1 homologues in rice

Flap endonuclease-1 (FEN-1) is an important enzyme involved in DNA replication and repair. Previously, we isolated and characterized a complementary DNA (cDNA) from rice (Oryza sativa) encoding a protein which shows homology with the eukaryotic flap endonuclease-1 (FEN-1). In this report, we found that rice (O. sativa L. cv. Nipponbare) possessed two FEN-1 homologues designated as OsFEN-1a and OsFEN-1b. The OsFEN-1a and OsFEN-1b genes were mapped to chromosome 5 and 3, respectively. Both genes contained 17 exons and 16 introns. Alignment of OsFEN-1a protein with OsFEN-1b protein showed a high degree of sequence similarity, particularly around the N and I domains. Northern hybridization and in situ hybridization analysis demonstrated preferential expression of OsFEN-1a and OsFEN-1b in proliferating tissues such as the shoot apical meristem or young leaves. The levels of OsFEN-1a and OsFEN-1b expression were significantly reduced when cell proliferation was temporarily halted by the removal of sucrose from the growth medium. When the growth-halted cells began to regrow following the addition of sucrose to the medium, both OsFEN-1a and OsFEN-1b were again expressed at high level. These results suggested that OsFEN-1a and OsFEN-1b are required for cell proliferation. Functional complementation assay suggested that OsFEN-1a cDNA had the ability to complement Saccharomyces cerevisiae rad27 null mutant. On the other hand, OsFEN-1b cDNA could not complement the rad27 mutant. The roles of OsFEN-1a and OsFEN-1b in plant DNA replication and repair are discussed.

A plant homologue of 36 kDa subunit of replication factor C: molecular cloning and characterization

Abstract We have isolated a 1.47 kb cDNA from rice (Oryza sativa) encoding plant homologue of a small subunit of Replication Factor C (RFC), and designated OsRFC3. The open reading frame of OsRFC3 encoded a predicted product of 367 amino acid residues with a molecular mass of 40.3 kDa. Genomic analysis indicated that the subunit existed as a single copy per genome. The transcript was highly expressed in root tips that contain root apical meristem, and the young and mature leaves. The mRNA level of OsRFC3 was significantly reduced when the cell proliferation was temporarily halted by removal of sucrose from the growth medium. When the growth-halted cells began to re-grow following addition of sucrose to the medium, OsRFC3 was again expressed at high level. The roles of OsRFC3 in plant DNA replication is discussed.

Arabidopsis DNA polymerase lambda mutant is mildly sensitive to DNA double strand breaks but defective in integration of a transgene.

The DNA double-strand break (DSB) is a critical type of damage, and can be induced by both endogenous sources (e.g., errors of oxidative metabolism, transposable elements, programmed meiotic breaks, or perturbation of the DNA replication fork) and exogenous sources (e.g., ionizing radiation or radiomimetic chemicals). Although higher plants, like mammals, are thought to preferentially repair DSBs via nonhomologous end joining (NHEJ), much remains unclear about plant DSB repair pathways. Our reverse genetic approach suggests that DNA polymerase λ is involved in DSB repair in Arabidopsis. The Arabidopsis T-DNA insertion mutant (atpolλ-1) displayed sensitivity to both gamma-irradiation and treatment with radiomimetic reagents, but not to other DNA damaging treatments. The atpolλ-1 mutant showed a moderate sensitivity to DSBs, while Arabidopsis Ku70 and DNA ligase 4 mutants (atku70-3 and atlig4-2), both of which play critical roles in NHEJ, exhibited a hypersensitivity to these treatments. The atpolλ-1/atlig4-2 double mutant exhibited a higher sensitivity to DSBs than each single mutant, but the atku70/atpolλ-1 showed similar sensitivity to the atku70-3 mutant. We showed that transcription of the DNA ligase 1, DNA ligase 6, and Wee1 genes was quickly induced by BLM in several NHEJ deficient mutants in contrast to wild-type. Finally, the T-DNA transformation efficiency dropped in NHEJ deficient mutants and the lowest transformation efficiency was scored in the atpolλ-1/atlig4-2 double mutant. These results imply that AtPolλ is involved in both DSB repair and DNA damage response pathway.