Subject(s)
DNA Repair/genetics , DNA Replication/genetics , DNA, Plant/genetics , Plants/genetics , Plastids/genetics , DNA Helicases/physiology , DNA Primase/physiology , DNA Topoisomerases, Type I/physiology , DNA-Directed DNA Polymerase/physiology , Rec A Recombinases/physiology , Replication Protein A/physiologyABSTRACT
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.
Subject(s)
DNA-Directed DNA Polymerase/genetics , Oryza/enzymology , Plastids/enzymology , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Conserved Sequence , DNA Primers , DNA Repair , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/metabolism , Kinetics , Molecular Sequence Data , Oryza/genetics , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Polymerase Chain Reaction , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
Adenosine Triphosphatases/genetics , DNA Helicases/genetics , Oryza/genetics , Plant Proteins/genetics , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Bleomycin/toxicity , Cloning, Molecular , DNA Damage , DNA Helicases/metabolism , DNA Repair , Gene Expression Profiling , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/radiation effects , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/radiation effects , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/radiation effects , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hydrogen Peroxide/toxicity , In Situ Hybridization , Isoenzymes/genetics , Isoenzymes/metabolism , Methyl Methanesulfonate/toxicity , Microscopy, Fluorescence , Molecular Sequence Data , Oryza/enzymology , Oryza/growth & development , Phylogeny , Plant Proteins/metabolism , RecQ Helicases , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
Arabidopsis/genetics , Arabidopsis/metabolism , DNA Polymerase I/genetics , DNA Polymerase I/metabolism , Plastids/genetics , Plastids/metabolism , Amino Acid Sequence , DNA Polymerase I/chemistry , Molecular Sequence Data , Plant Structures/genetics , Plant Structures/metabolism , Sequence Homology, Amino Acid , Tissue DistributionABSTRACT
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.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , DNA Damage/physiology , Oryza/genetics , Oryza/metabolism , Subcellular Fractions/metabolism , Amino Acid Sequence , Cells, Cultured , Gene Expression Regulation/physiology , Molecular Sequence Data , Molecular Weight , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Tissue DistributionABSTRACT
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.
