Plant DNA Replication Research Articles

Characterization of the origin recognition complex (ORC) from a higher plant, rice ( Oryza sativa L.)

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.

Plant DNA Topoisomerases: Structure, Function, and Cellular Roles in Plant Development

Regulation of the topological state of DNA is critical for cellular viability. DNA topoisomerases alter DNA topology and participate in nearly all events related to DNA metabolism such as replication, transcription, recombination, and chromosome segregation. Despite significant progress in our understanding of the structure, mechanism, and the role of topoisomerases in diverse cellular processes in bacteria, yeast, and animal systems, the roles of plant topoisomerases are poorly understood. In recent years expression studies, characterization of recombinant topoisomerases, and gene knockout studies have highlighted the importance of topoisomerases in plant DNA replication, mitosis, cell proliferation, organelle division, meiotic recombination, morphogenesis, and plant development. We have focused this review specifically on plant topoisomerases, with an emphasis on their biological role in plant development.

The DNA-A component of a plant geminivirus (Indian mung bean yellow mosaic virus) replicates in budding yeast cells.

Understanding the biochemistry of DNA replication of the plant DNA viruses is important for the development of antiviral strategies. Since DNA replication is little studied in plants, a genetically tractable, easily culturable, eukaryotic model system is required to pursue such studies in a facile manner. Here we report the development of a yeast model system that supports DNA replication of a chosen geminivirus strain, Indian mung bean yellow mosaic virus. The replication of plasmid DNA in the model system relies specifically on the virus-derived elements and factors. Usage of this model system revealed the role of at least one hitherto unknown viral factor for viral DNA replication. The episomal characteristic of single-strandedness of replicated plasmid DNA was shown, and the expression of viral genes was also confirmed. This model system is expected to shed light on the machinery and mechanism involved in geminiviral DNA replication in plants.

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.

DNA replication in plants: characterization of a cdc6 homologue from Arabidopsis thaliana.

Cdc6 is a key regulator of DNA replication in eukaryotes. In this work, the expression pattern of an Arabidopsis cdc6 homologue is characterized by RT-PCR and in situ hybridization. The data suggest that cdc6At expression is cell cycle regulated. During development, high cdc6At mRNA levels are found in regular cycling cells. In addition, cdc6At expression is also observed in cells that are probably undergoing endoreduplication, suggesting a possible role of Cdc6At in this process in plants.

Two types of replication protein A 70 kDa subunit in rice, Oryzasativa: 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 (Oryzasativa cv. Nipponbare) termed OsRPA70a and OsRPA32.OsRPA70a shows a low level of homology with OsRPA1 which was isolated from deepwater rice (Oryzasativa cv. Pin Gaew 56), previously. We also succeeded to isolate OsRPA70b which is homologue to OsRPA1 from Oryzasativa cv. Nipponbare. OsRPA70a shows only 33.8% sequence identity with OsRPA70b, indicating that two different types of 70 kDa RPA subunits are present in Oryzasativa 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.

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

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.

Molecular cloning and characterization of a plant homologue of the origin recognition complex 1 (ORC1)

By using the rice EST database, we have isolated a 2.8 kb cDNA, termed Oryza sativa ORC1 ( OsORC1), from rice ( O. sativa) encoding a protein that shows homology with the eukaryotic ORC1 proteins. Alignment of the OsORC1 protein sequence with the sequence of ORC1 from human and yeasts S. cerevisiae and S. pombe showed a high degree of sequence homology (38.7, 32.9 and 35.0% identity, respectively), particularly around the C-terminal region containing the CDC-NTP domain. Interestingly, the OsORC1 protein had an A+T hook-like motif, which was not present in the human or yeast genes. Genomic analysis indicated that OsORC1 existed as a single copy per genome. OsORC1 transcripts were expressed strongly in root tips and weakly in young leaves containing root apical meristem and marginal meristem, respectively. No expression was detected in the mature leaves. The level of OsORC1 expression was significantly reduced when cell proliferation was temporarily halted by the removal of sucrose from the growth medium. When the growth-halted cells began to re-grow following addition of sucrose to the medium, OsORC1 was again expressed at high levels. These results suggested that OsORC1 is required for cell proliferation. The role of OsORC1 in plant DNA replication will be discussed.

Subunit composition of DNA polymerases A and B from wheat cell

DNA polymerases A and B purified from wheat ( Triticum monococcum) embryos were previously shown to be respectively the plant counterparts of mammalian DNA polymerases α and δ. From wheat cultured cells, we isolated a protein fraction able to replicate a DNA template/primer in a cell-free DNA replication assay. This fraction contains the DNA polymerases pol A and pol B, exhibiting the same biochemical properties as those found in wheat embryo. The catalytic subunits of DNA polymerases pol A and B purified from this fraction were analysed by a DNA polymerase trap assay and their molecular mass were respectively determined as 90 and 125 kDa. This shows that pol A catalytic subunit is shorter than those of yeast or mammal DNA polymerases α (respectively 180 and 165 kDa), whereas pol B catalytic subunit exhibits the same molecular mass as yeast and mammal DNA polymerases δ (125 kDa). Catalytic subunit identification using DNA polymerase trap assay could be a good alternative to isolate and sequence active polypeptides from low purified enzymes. These results contribute to the molecular characterization of DNA replication enzymes in plants and will permit to establish a plant DNA replication model.

DNA replication and cell cycle in plants: learning from geminiviruses.

Plant cell growth and development depend on continuous cell proliferation which is restricted to small regions of the plant called meristems. Infection by geminiviruses, small DNA viruses whose replicative cycle relies on host cell factors, is excluded from those proliferating areas. Since most of the replicative factors are present, almost exclusively, in proliferating cells, geminivirus infection is believed to induce a cellular state permissive for viral DNA replication, e.g. S-phase or, at least, some specific S-phase functions. The molecular basis for this effect seems to be the interference that certain geminivirus proteins exert on the retinoblastoma-related (RBR) pathway, which analogously to that of animal cells, regulates plant cell cycle activation and G(1)-S transition. In some cases, geminiviruses induce cell proliferation and abnormal growth. Mechanisms other than sequestering plant RBR probably contribute to the multiple effects of geminivirus proteins on cellular gene expression, cell growth control and cellular DNA replication. Current efforts to understand the coupling of geminivirus DNA replication to cell cycle and growth control as well as the directions in which future research is aiming are reviewed.

Geminiviruses: Models for Plant DNA Replication, Transcription, and Cell Cycle Regulation

Geminiviruses have small, single-stranded DNA genomes that replicate through double-stranded intermediates in the nuclei of infected plant cells. Viral double-stranded DNA also assembles into minichromosomes and is transcribed in infected cells. Geminiviruses encode only a few proteins for their replication and transcription and rely on host enzymes for these processes. However, most plant cells, which have exited the cell cycle and undergone differentiation, do not contain the replicative enzymes necessary for viral DNA synthesis. To overcome this barrier, geminiviruses induce the accumulation of DNA replication machinery in mature plant cells, most likely by modifying cell cycle and transcriptional controls. In animals, several DNA viruses depend on host replication and transcription machinery and can alter their hosts to create an environment that facilitates efficient viral replication. Analysis of these viruses and their proteins has contributed significantly to our understanding of DNA replication, transcription, and cell cycle regulation in mammalian cells. Geminiviruses have the same potential for plant systems. Plants offer many advantages for these types of studies, including ease of transformation, well-defined cell populations and developmental programs, and greater tolerance of cell cycle perturbation and polyploidy. Our knowledge of the molecular and cellular events that mediate geminivirus infection has increased significantly during recent years. The goal of this review is to summarize recent research addressing geminivirus DNA replication and its integration with transcriptional and cell cycle regulatory processes.

Geminiviruses: Models for Plant DNA Replication, Transcription, and Cell Cycle Regulation

Geminiviruses have small, single-stranded DNA genomes that replicate through double-stranded intermediates in the nuclei of infected plant cells. Viral double-stranded DNA also assembles into minichromosomes and is transcribed in infected cells. Geminiviruses encode only a few proteins for their replication and transcription and rely on host enzymes for these processes. However, most plant cells, which have exited the cell cycle and undergone differentiation, do not contain the replicative enzymes necessary for viral DNA synthesis. To overcome this barrier, geminiviruses induce the accumulation of DNA replication machinery in mature plant cells, most likely by modifying cell cycle and transcriptional controls. In animals, several DNA viruses depend on host replication and transcription machinery and can alter their hosts to create an environment that facilitates efficient viral replication. Analysis of these viruses and their proteins has contributed significantly to our understanding of DNA replication, transcription, and cell cycle regulation in mammalian cells. Geminiviruses have the same potential for plant systems. Plants offer many advantages for these types of studies, including ease of transformation, well-defined cell populations and developmental programs, and greater tolerance of cell cycle perturbation and polyploidy. Our knowledge of the molecular and cellular events that mediate geminivirus infection has increased significantly during recent years. The goal of this review is to summarize recent research addressing geminivirus DNA replication and its integration with transcriptional and cell cycle regulatory processes.

An ATP-inhibited endonuclease from cauliflower ( Brassica oleracea var. botrytis ) inflorescence: purification and characterization

In our studies on the role of enzymes in plant DNA replication, recombination, and repair, we isolated from cauliflower (Brassica oleracea L. var. botrytis) inflorescences a single-stranded DNA-specific endonuclease that was inhibited by ATP. The endonuclease, designated cauliflower nuclease II, was purified to near homogeneity through six successive column chromatographies. The enzyme is a single polypeptide with a molecular mass of 70 kDa as judged by the results of sodium dodecyl sulfate-polyacry amide gel electrophoresis, activity gel, and gel-filtration column chromatography. The enzyme can cleave a linear or a circular single-stranded DNA but cannot cut or nick a double-stranded DNA. The mode of activity of the nuclease is endonucleolytic and non-processive. Interestingly, the endonuclease activity is strongly inhibited by less than 0.1 mM ATP, although the role of this inhibition is thus far unclear. While ATPγS and GTP can also inhibit the activity, other ribonucleoside triphosphates are much less effective. The optimum pH of the enzyme is 5.6. The enzyme requires an exceptionally high ionic strength, 0.2 M KCI for optimum activity, and without these ions no activity can be detected. The endonuclease activity is stimulated by Ca2+, which cannot be replaced by Mg2+ or Mn2+. The features of the enzyme and its relation to plant DNA metabolism are discussed.

Molecular cloning of the cDNA for the catalytic subunit of plant DNA polymerase alpha and its cell-cycle dependent expression.

DNA polymerase alpha has been studied in considerable detail in yeast and animals. Genetic and biochemical analyses reveal that this enzyme is composed of a heterotetramer and is necessary for replicon initiation and primer synthesis in lagging strand synthesis. In spite of the fact that modes of DNA replication in plants seem to be similar to those in other eukaryotes, very little is known about the biochemical components that participate in DNA replication of plants, including DNA polymerases. Using a 561-base pair DNA fragment, obtained by polymerase chain reaction amplification from a rice cDNA library as a probe, we isolated and sequenced a cDNA homologous to the cDNA for the catalytic subunit of rice DNA polymerase alpha. The encoded polypeptide has extensive homology with the catalytic subunit of DNA polymerase alpha from several species. Furthermore, when the cDNA was expressed in eukaryotic transcription/translation systems, the protein products showed DNA polymerase activity which was inhibited by a monoclonal antibody specific for DNA polymerase alpha. Using RNA gel blot analysis, we found that the levels of mRNA of the catalytic subunit of this enzyme is regulated during the cell-cycle in plant cells. This is the first report which describes the cDNA cloning of plant DNA polymerase. We conclude that the principal features of the DNA polymerase alpha catalytic subunit are conserved in plants.

Mutational analysis of potato yellow mosaic geminivirus.

Mutations have been inserted into the virion and complementary sense ORFs encoding proteins with M(r)s in excess of 9 kDa of both DNA A and DNA B of potato yellow mosaic geminivirus (PYMV). Wild-type and mutant monomeric clones were tested for their ability to replicate, produce PYMV-specific DNA, spread and cause symptoms in Nicotiana benthamiana plants following biolistic inoculation. Dimeric clones of the DNA A mutants were also investigated by agroinoculation of leaf discs. In contrast to N. benthamiana plants agroinoculated with PYMV DNA A, in which the wild-type DNA A component was capable of limited independent replication and spread, both excised DNA A and B components were required for DNA replication and symptom development in plants inoculated by the biolistic method. Mixtures of both genomic components were also infectious for potato plants following biolistic inoculation. Mutations in ORFs AL1, AL2, BR1 and BL1 resulted in clones incapable of infecting N. benthamiana plants. However, the AL2 mutation, but not the AL1 mutation, allowed viral DNA replication in leaf discs. Mutations to both the AR1 and AL3 ORFs produced clones which were infectious in plants but showed a considerable delay in the production of attenuated symptoms as compared to wild-type infections. Mutating the AL3 ORF dramatically reduced viral DNA replication in both whole plants and leaf discs. Mutations to the AL4 ORF produced clones which were as infectious for both N. benthamiana and potato plants as the wild-type clones. Our results are compared with those from mutagenesis studies on related bipartite geminiviruses.

Wheat DNA Primase (RNA Primer Synthesis in Vitro, Structural Studies by Photochemical Cross-Linking, and Modulation of Primase Activity by DNA Polymerases).

DNA primase synthesizes short RNA primers used by DNA polymerases to initiate DNA synthesis. Two proteins of approximately 60 and 50 kD were recognized by specific antibodies raised against yeast primase subunits, suggesting a high degree of analogy between wheat and yeast primase subunits. Gel-filtration chromatography of wheat primase showed two active forms of 60 and 110 to 120 kD. Ultraviolet-induced cross-linking with radioactive oligothymidilate revealed a highly labeled protein of 60 kD. After limited trypsin digestion of wheat (Triticum aestivum L.) primase, a major band of 48 kD and two minor bands of 38 and 17 kD were observed. In the absence of DNA polymerases, the purified primase synthesizes long RNA products. The size of the RNA product synthesized by wheat primase is considerably reduced by the presence of DNA polymerases, suggesting a modulatory effect of the association between these two enzymes. Lowering the primase concentration in the assay also favored short RNA primer synthesis. Several properties of the wheat DNA primase using oligoadenylate [oligo(rA)]-primed or unprimed polythymidilate templates were studied. The ability of wheat primase, without DNA polymerases, to elongate an oligo(rA) primer to long RNA products depends on the primer size, temperature, and the divalent cation concentration. Thus, Mn2+ ions led to long RNA products in a very wide range of concentrations, whereas with Mg2+ long products were observed around 15 mM. We studied the ability of purified wheat DNA polymerases to initiate DNA synthesis from an RNA primer: wheat DNA polymerase A showed the highest activity, followed by DNA polymerases B and CII, whereas DNA polymerase CI was unable to initiate DNA synthesis from an RNA primer. Results are discussed in terms of understanding the role of these polymerases in DNA replication in plants.

DNA sequences essential for replication of the B genome component of tomato golden mosaic virus.

The genome of the geminivirus tomato golden mosaic virus (TGMV) is divided between two DNA components, designated A and B, which differ in sequence except for a 230-nucleotide common region. The A genome component is known to encode viral functions necessary for viral DNA replication, while the B genome component specifies functions necessary for spread of the virus through the infected plant. To identify cis-acting sequences required for viral DNA replication, several mutants were constructed by the introduction of small insertions into TGMV B at selected sites within and just outside the common region. Other mutants had the common region inverted or deleted. All of the mutants were tested for their effects on infectivity and DNA replication in whole plants and leaf discs. Our results indicate that the common region in its correct orientation is required for infectivity and for replication of TGMV B. Furthermore, the conserved hairpin loop sequence located within the TGMV common region and found in all geminiviruses is necessary for DNA replication, and may be part of the viral replication origin.

Immunocytochemical Detection of DNA Synthesis in Plant Cells

Summary An immunocytochemical protocol has been developed to detect DNA synthesis in plant protoplasts and cells using soybean as a model system. Cells were labelled with the thymidine analog, 5-bromo-2deoxyuridine (BrdU) and fixed for antibody treatments. Formaldehyde fixation gave better results than a mixture of methanol and acetic acid. Suspension cells required an enzyme treatment to partially remove the cell wall. Fixed protoplasts and cells were extracted with Triton X-100 and reacted with a monoclonal antibody against BrdU. The BrdU labelled nuclei were visualized with either enzyme- or fluorescein-conjugated secondary antibodies. Aphidicolin reduced the number of nuclei labelled by more than 50 % at lopg-ml-', inhibited the labelling completely at 501g- ml-', confirming that the labelling was due to replicative nuclear DNA synthesis. Immunocytochemistry is simpler than autoradiography and allows simultaneous detection of DNA and other antigens including, for example, microtubules. This technique should be widely useful in studies of plant DNA replication.

Enzymology of nuclear dna replication in plants

DNA replication is a complicated, multi‐step process and must therefore involve an array of different enzyme activities. These enzymes may be loosely associated together to form a replication complex. A number of different enzymes which are assumed to be involved in DNA replication have been identified. These include endodeoxyribonuclease, topoisomerase, “primase”, DNA polymerase, ribonuclease‐H, DNA ligase, and DNa methylase. At least two of these enzymes, DNA polymerase and DNA ligase, exist as multiple forms. The best characterized of the enzymes is DNA polymerase, where it has been possible to assign a role in DNA replication to one specific form, polymerase‐α. With DNA polymerase‐α, there is also some information on regulatory mechanisms, and on subunit composition. By contrast, others of the enzymes listed above, despite partial characterization, have not yet been identified as having a specific function in replication. Further progress will be made by a number of approaches, including further characterization of the enzymes, use of conditional mutants, developmental studies, and in vitro reconstructions using defined template‐primers and purified enzymes.

Differential DNA Replication in Plants: A Critical Review

Summary Differential DNA replication is a controversal item of plant physiology and cell biology. While some authors envisage such events as basic mechanisms of cell differentiation and morphogenesis, others consider them as pathological slips or even methodical errors. This critical review may help to realize the facts and questions, and to find out the advantage and disadvantage of the methods used at present. New evidence of differential DNA replication is reported, and some suggestions concernig methodical improvements are made. Although the occurrence of somatic DNA amplification and DNA underreplication is very likely, it cannot be proven unequivocally with the present-day techniques, and thus its physiological significance remains speculative. It is emphasized that more data should be gathered using improved methods, and that experimental systems should be elaborated which allow to analyze the possible causal relationship between differential DNA replication and cell differentiation and morphogenesis.