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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.

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.

The origin recognition complex (ORC) protein plays a critical role in DNA replication through binding to sites (origins) where replication commences. The protein is composed of six subunits (ORC1 to 6) in animals and yeasts. Our knowledge of the ORC protein in plants is, however, much less complete. We have performed cDNA cloning and characterization of ORC subunits in rice (Oryza sativa L. cv. Nipponbare) in order to facilitate study of plant DNA replication mechanisms. Our previous report provided a description of a gene, ORC1 (OsORC1), that encodes one of the protein subunits. The present report extends this initial analysis to include the genes that encode four other rice ORC subunits, OsORC2, 3, 4 and 5. Northern hybridization analyses demonstrated the presence of abundant transcripts for all OsORC subunits in shoot apical meristems (SAM) and cultured cells, but not in mature leaves. Interestingly, only OsORC5 showed high levels of expression in organs in which cell proliferation is not active, such as flag leaves, the ears and the non-tip roots. The pattern of expression of OsORC2 also differed from other OsORC subunits. When cell proliferation was temporarily halted for 6-10 days by removal of sucrose from the growth medium, expression of OsORC1, OsORC3, OsORC4 and OsORC5 was substantially reduced. However, the level of expression of OsORC2 remained constant. We suggest from these results that expression of OsORC1, 3, 4 and 5 are correlated with cell proliferation, but the expression of OsORC2 is not.

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.

Little is known about the functions of DNA polymerase lambda (Pol lambda) recently identified in mammals. From the genomic sequence information of rice and Arabidopsis, we found that Pol lambda may be the only member of the X-family in higher plants. We have succeeded in isolating the cDNA and recombinant protein of Pol lambda in a higher plant, rice (Oryza sativa L. cv. Nipponbare) (OsPol lambda). OsPol lambda had activities of DNA polymerase, terminal deoxyribonucleotidyl transferase and deoxyribose phosphate lyase, a marker enzyme for base excision repair. It also interacted with rice proliferating cell nuclear antigen (OsPCNA) in a pull-down assay. OsPCNA increased the processivity of OsPol lambda. Northern blot analysis showed that the level of OsPol lambda expression correlated with cell proliferation in meristematic and meiotic tissues, and was induced by DNA-damaging treatments. These properties suggest that plant Pol lambda is a DNA repair enzyme which functions in plant meristematic and meiotic tissues, and that it can substitute for Pol beta and terminal deoxyribonucleotidyl transferase.

Calreticulin (CRT), a major Ca2+ -sequestering protein, has been implicated in a variety of cellular functions such as Ca2+ storage, signaling and chaperone activity within the cytoplasm and endoplasmic reticulum. To investigate the biological role of CRT in rice, 21 partial cDNAs, encoding proteins that interacted with rice CRT in a yeast two-hybrid interaction-cloning system, were characterized and the nucleotide sequences were found to be identical to each other. A full-length cDNA of 3.5 kb, obtained from rice genomic sequence data and 5' RACE, codes for a novel protein of 966 amino acid residues and was designated as CRTintP (CRT interacting protein). Primary sequence analysis of CRTintP showed no sequence homology with the known functional proteins; however, a potential ubiquitin-like domain at the N-terminal together with a putative leucine zipper, a nuclear localization signal and several sites for serine/threonine kinases were evident. Cellular localization of CRTintP demonstrated its role in directing green fluorescent protein to the nucleus in onion epidermal cells. Northern and immunoblot analysis showed increased expression of CRT and CRTintP in response to cold stress. Co-immunoprecipitation using anti-CRT antibodies confirmed the existence of the CRT-CRTintP complex in vivo in the stressed leaf tissue, suggesting their potential role in regulating stress response.

We report here the existence of interactions between a ubiquitin-conjugating enzyme, Rad6, from rice, Oryza sativa L. cv. Nipponbare (OsRad6), and Sgt1 (OsSgt1), a novel subunit of the SCF ubiquitin ligase complex. Rad6 is not only related to post-replicational repair but also to the proteasome system, while Sgt1 has a function in kinetochore assembly. The relationship between the two is unexpected, but of great interest. The open reading frames of OsRad6 and OsSgt1 encode predicted products of 152 and 367 amino acid residues, respectively, with molecular weights of 17.3 and 40.9kDa. Two-hybrid and pull-down analyses indicated that OsRad6 binds to OsSgt1, and transcripts of both OsRad6 and OsSgt1 were found to be strongly expressed only in the proliferating tissues such as the shoot apical meristem, suggesting that their expression is cell cycle-dependent. The amount of the Rad6 mRNA in cultured cells increased rapidly after division was halted, and mRNA levels of Rad6 and Sgt1 were induced by UV- and DNA-damaging agents such as MMS or H(2)O(2). The Rad6 pathway for repair or the proteasome system may thus require Sgt1 as ubiquitin-conjugating enzyme.

To study whether metabolic control of proliferating cell nuclear antigen (PCNA) during the cell cycle is similar to that of associated protein factors, two-hybrid analysis with PCNA from rice (Oryza sativa L. cv. Nipponbare) was performed. PCNA interacted with rice Rpt6, which is the ATPase subunit of 26S proteasome, both in vitro and in vivo, and the degradation of PCNA was disrupted by the proteasome in vivo. The tissue-specific expression pattern of the transcripts of Rpt6 and PCNA suggested that the rice proteasome played important roles in DNA replication involving PCNA. These findings indicate a proteasome-dependent degradation of PCNA.

The eukaryotic polymerase processivity factor, proliferating cell nuclear antigen (PCNA), interacts with many cell cycle-regulator proteins and with proteins involved in the mechanisms of DNA replication and repair. In the present study using two-hybrid analysis with PCNA from rice, Oryza sativa L. cv. Nipponbare (OsPCNA), we found that OsPCNA interacted in vitro and in vivo with rice JUN-activation-domain-binding protein 1 (OsJab1), which is known as COP9/signalsome subunit 5. Both OsPCNA and OsJab1 transcripts were expressed strongly in the shoot apical meristem and weakly in young leaves, flag leaves, ears, roots and root tips. No expression was detected in the mature leaves. The OsPCNA and OsJab1 proteins were expressed and accumulated mostly in the shoot apical meristem and ears, suggesting that OsJab1 is involved in cell proliferation in cooperation with OsPCNA. The role of OsPCNA with OsJab1 in plant DNA proliferation is discussed.

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.

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.