RESUMO
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.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Oryza/genética , Mapeamento Cromossômico , Clonagem Molecular , DNA Polimerase I/genética , DNA Complementar/química , DNA Complementar/genética , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/enzimologia , Éxons/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Hibridização In Situ , Dados de Sequência Molecular , Oryza/enzimologia , Oryza/crescimento & desenvolvimento , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Caules de Planta/enzimologia , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Análise de Sequência de DNAAssuntos
Reparo do DNA/genética , Replicação do DNA/genética , DNA de Plantas/genética , Plantas/genética , Plastídeos/genética , DNA Helicases/fisiologia , DNA Primase/fisiologia , DNA Topoisomerases Tipo I/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Recombinases Rec A/fisiologia , Proteína de Replicação A/fisiologiaRESUMO
Plastids are organelles unique to plant cells and are responsible for photosynthesis and other metabolic functions. Despite their important cellular roles, relatively little is known about the mechanism of plastidial DNA replication and repair. Recently, we identified a novel DNA polymerase in Oryza Sativa L. (OsPOLP1, formerly termed OsPolI-like) that is homologous to prokaryotic DNA polymerase Is (PolIs), and suggested that this polymerase might be involved in plastidial DNA replication and repair. Here, we propose to rename the plant PolI homologs as DNA polymerase pi (POLP), and investigate the biochemical properties of full-length OsPOLP1. The purified OsPOLP1 elongated both DNA and RNA primer hybridized to a DNA template, and possessed a 3' exonuclease activity. Moreover, OsPOLP1 displayed high processivity and fidelity, indicating that this polymerase has the biochemical characteristics appropriate for DNA replication. We found that POLPs have two extra sequences in the polymerase domain that are absent in prokaryotic PolIs. Deletion of either insert from OsPOLP1 caused a decrease in DNA synthetic activity, processivity, and DNA binding activity. In addition, OsPOLP1 efficiently catalyzed strand displacement on nicked DNA with a 5'-deoxyribose phosphate, suggesting that this enzyme might be involved in a repair pathway similar to long-patch base excision repair. These results provide insights into the possible role of POLPs in plastidial DNA replication and repair.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Oryza/enzimologia , Plastídeos/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência Conservada , Primers do DNA , Reparo do DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Cinética , Dados de Sequência Molecular , Oryza/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Adenosina Trifosfatases/genética , DNA Helicases/genética , Oryza/genética , Proteínas de Plantas/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Bleomicina/toxicidade , Clonagem Molecular , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Peróxido de Hidrogênio/toxicidade , Hibridização In Situ , Isoenzimas/genética , Isoenzimas/metabolismo , Metanossulfonato de Metila/toxicidade , Microscopia de Fluorescência , Dados de Sequência Molecular , Oryza/enzimologia , Oryza/crescimento & desenvolvimento , Filogenia , Proteínas de Plantas/metabolismo , RecQ Helicases , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
We isolated and characterized the rice homologue of the DNA repair gene Snm1 (OsSnm1). The length of the cDNA was 1862bp; the open reading frame encoded a predicted product of 485 amino acid residues with a molecular mass of 53.2kDa. The OsSnm1 protein contained the conserved beta-lactamase domain in its internal region. OsSnm1 was expressed in all rice organs. The expression was induced by MMS, H(2)O(2), and mitomycin C, but not by UV. Transient expression of an OsSnm1/GFP fusion protein in onion epidermal cells revealed the localization of OsSnm1 to the nucleus. These results suggest that OsSnm1 is involved not only in the repair of DNA interstrand crosslinks, but also in various other DNA repair pathways.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dano ao DNA/fisiologia , Oryza/genética , Oryza/metabolismo , Frações Subcelulares/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Previously, we described a novel DNA polymerase, designated as OsPolI-like, from rice. The OsPolI-like showed a high degree of sequence homology with the DNA polymerase I of cyanobacteria and was localized in the plastid. Here, we describe two PolI-like polymerases, designated as AtPolI-like A and AtPolI-like B, from Arabidopsis thaliana. In situ hybridization analysis demonstrated expression of both mRNAs in proliferating tissues such as the shoot apical meristem. Analysis of the localizations of GFP fusion proteins showed that AtPolI-like A and AtPolI-like B were localized to plastids. AtPolI-like B expression could be induced by exposure to the mutagen H(2)O(2). These results suggested that AtPolI-like B has a role in the repair of oxidation-induced DNA damage. Our data indicate that higher plants possess two plastid DNA polymerases that are not found in animals and yeasts.