DNA polymerases from plant cells

Litvak, Simón; Catroviejo, Michel

doi:10.1016/0027-5107(87)90290-9

Cited by 22 publications

(11 citation statements)

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“…In order to settle this dilemma, the effects of known inhibitors of in vitro ligation were tested. Aphidicolin (inhibitor of DNA polymerases ␣, ␦, and ε [5,14,34]) and ddTTP (inhibitor of DNA polymerases ␤ and ␥ [17]) did not inhibit the reaction (Fig. 3D).…”

Section: Resultsmentioning

confidence: 96%

Plant Enzymes but Not Agrobacterium VirD2 Mediate T-DNA Ligation In Vitro

Ziemienowicz

Tinland

Bryant

et al. 2000

Molecular and Cellular Biology

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Agrobacterium tumefaciens, a gram-negative soil bacterium, transfers DNA to many plant species. In the plant cell, the transferred DNA (T-DNA) is integrated into the genome. An in vitro ligation-integration assay has been designed to investigate the mechanism of T-DNA ligation and the factors involved in this process. The VirD2 protein, which is produced in Agrobacterium and is covalently attached to T-DNA, did not, under our assay conditions, ligate T-DNA to a model target sequence in vitro. We tested whether plant extracts could ligate T-DNA to target oligonucleotides in our test system. The in vitro ligation-integration reaction did indeed take place in the presence of plant extracts. This reaction was inhibited by dTTP, indicating involvement of a plant DNA ligase. We found that prokaryotic DNA ligases could substitute for plant extracts in this reaction. Ligation of the VirD2-bound oligonucleotide to the target sequence mediated by T4 DNA ligase was less efficient than ligation of a free oligonucleotide to the target. T-DNA ligation mediated by a plant enzyme(s) or T4 DNA ligase requires ATP.The soil bacterium Agrobacterium tumefaciens is a plant pathogen responsible for tumor induction on dicotyledonous plants through its ability to transfer DNA carrying plant-active oncogenes to the plant cell (16,25,36). Because of this ability, Agrobacterium is widely used for plant transformation (12). The transferred DNA (T-DNA), which in Agrobacterium resides on a large tumor-inducing (Ti) plasmid, is processed within the bacterium and is exported to the plant, where it is integrated into the plant genome (28,29,30). Proteins encoded by the virulence (vir) region of the Ti plasmid regulate T-DNA processing and transfer. Virulence proteins recognize 24-bplong imperfect direct repeats (border sequences) that define the T-DNA. In the presence of the VirD1 protein, VirD2 cleaves the border sequence in a site-and strand-specific manner and concomitantly becomes covalently attached to the 5Ј end of the nicked DNA (9, 13, 33, 35). The nicked DNA is then displaced 5Ј to 3Ј from the plasmid, producing single-stranded T-DNA (8). The T-DNA-VirD2 complex and the VirE2 protein are believed to be transferred to the plant with the help of a pilus-like structure containing the VirB and VirD4 proteins (2, 4, 10, 37). In the plant cell, T-DNA is coated with the single-stranded DNA (ssDNA)-binding protein VirE2 (8, 25), forming a T-DNA-protein complex that is imported into the nucleus, where the T-DNA is integrated into the nuclear genome. The VirD2 protein is transferred into the nucleus in conjunction with the T-DNA; it presumably remains attached to it up to the integration step.In higher eukaryotic organisms, such as plants, illegitimate recombination is the predominant mechanism of integration for naked DNA (20,22,24,26). Likewise, the T-DNA is integrated into the plant genome by illegitimate recombination, a mechanism in which two DNA molecules that do not share extensive homology are joined (11,18,19,31). It is not clear yet whe...

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Section: Resultsmentioning

confidence: 96%

Plant Enzymes but Not Agrobacterium VirD2 Mediate T-DNA Ligation In Vitro

Ziemienowicz

Tinland

Bryant

et al. 2000

Molecular and Cellular Biology

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“…In plants, a-, f3-, and y-like DNA polymerases have been identified (for a review, see ref. 29). There is no report as to the presence of a 8-like polymerase in plants; however, an a-like DNA polymerase isolated from wheat embryo has been shown to have 3'-5' exonuclease activity (30) as does the mammalian DNA polymerase 8 (3)(4)(5) …”

Section: Resultsmentioning

confidence: 99%

Gene for proliferating-cell nuclear antigen (DNA polymerase delta auxiliary protein) is present in both mammalian and higher plant genomes.

Suzuka

Daidoji

Matsuoka

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

134

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Proliferating-cell nuclear antigen (PCNA; also called cyclin) was originally described in proliferating mammalian cells as a nuclear protein with an apparent Mr of 33,000-36,000 and recently was found to be a DNA polymerase 6 auxiliary protein.To elucidate whether PCNA/cyclin is a universal protein necessary for proliferation of eukaryotes, a search was conducted for PCNA/cyclin homologues in higher plants. In Southern blot-hybridization analysis, a rat PCNA/ cyclin cDNA probe hybridized with homologous sequences in genomic DNAs from rice, soybean, and tobacco. A PCNA/ cyclin-related molecular clone (pCJ-1) was isolated from rice DNA and was partially sequenced. The pCJ-1 probe hybridized with a 1.2-kilobase transcript in RNA from rice root tips and shoots. Immunoblot analysis of the soluble extract of soybean root tips with monospecific anti-PCNA/cycin identified an immunoreactive protein with an apparent Mr of 34,000.Immunohistochemical analysis revealed the presence of an immunoreactive PCNA/cyclin protein in the nuclei of cells in the meristem of soybean root tips. The highly homologous nature of the gene for PCNA/cyclin throughout the animal and plant kingdoms suggests that the product of the gene plays an essential role in DNA replication in eukaryotes.Proliferating-cell nuclear antigen (PCNA; also called cyclin) has received considerable attention in recent years because it has been demonstrated to be identical to DNA polymerase 8 auxiliary protein (1, 2), which is required for the polymerase to copy templates such as poly(dA)/oligo(dT), primed M13 DNA, and denatured calf thymus DNA. DNA polymerase 8 (3-5) differs from other mammalian DNA polymerases such as DNA polymerases a, f3, and y by having an exonuclease activity. Since the exonuclease activity is in the 3'-5' direction, DNA polymerase 8 has a proofreading activity (6), as seen with the prokaryotic DNA polymerases (7), and is expected to play a significant role in the maintenance of the fidelity of mammalian DNA replication (6). From the studies on molecularly cloned cDNA for PCNA/cyclin (8-10) and on the antigenicity of PCNA/cyclin (11,12), it has been recognized that this protein may be similar among a wide range of higher eukaryotes and, thus, conserved during evolution. The deduced amino acid sequences from rat (8, 9) and human (10) PCNA/cyclin cDNAs reveal only 4 amino acid substi- MATERIALS AND METHODS Plant Materials. Two rice subspecies (Indica and Japonica of Oriza sativa L.), soybean (Glycine max Merril), and tobacco (Nicotiana tabacum L.) were used. The cultivars of Japonica rice used were Musashikogane, Taichung 65, Nipponbare, and Sasanishiki.Labeling of DNA Probe. The rat PCNA/cyclin cDNA, pCR-1 (8, 9), was used. The Pst I digest of the pCR-1 insert yielded two bands in addition to the plasmid DNA in gel electrophoresis. The larger fragment (0.75 kb) contains 88% of the coding region of rat PCNA/cyclin cDNA and was used as a hybridization probe (pCR-1 probe). The probe was radiolabeled to a specific activity of 1 x 109 cpm/,ug...

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“…DNA polymerases have also been isolated and characterized in plant cells (Bryant, 1980;Litvak and Castroviejo, 1987). Enzymes with similar properties to DNA polymerase a have been studied in periwinkle (Gardner and Kado, 1976), sugar beet (Tymonko and Dunham, 1977), rice (Amileni et al, 1979), spinach (Misumi and Weissbach, 1982), pea (Bryant et al, 1992), wheat (Laquel et al, 1990;Balmukhanov et al, 1992), and maize (Zen mnys L.) (Coello et al, 1992).…”

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confidence: 99%

Studies on the Processivity of Maize DNA Polymerase 2, an [alpha]-Type Enzyme

Coello

Vázquez‐Ramos

1995

Plant Physiol.

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This paper describes studies on the processivity of an a-type DNA polymerase from maize (Zea mays L.) embryonic axes, designated as DNA polymerase 2. Using poly(dA)/oligo(dT) as template, DNA polymerase 2 has a processivity of 18 (25) nucleotides incorporated, a value much lower than that found for wheat a-type DNA polymerase (P. Laquel, S. Litvak, M. Castroviejo [I9931 Plant Physiol 102: 107-1 14). Conditions that maximally stimulate enzyme activity, such as 1 O0 mM KCI and 12 mM MgZ+, are strongly inhibitory of processivity and cause the enzyme to become distributive under these conditions. Optimal concentrations for processivity are 1 O mM KCI and 1 to 2 mM Mg2+. Both enzyme activity and processivity were found to be similar at different MnZ+ concentrations. Both DNA polymerase 2 activity and processivity are greatly reduced by spermine and Kethylmaleimide. A distinguishing feature of processivity in DNA polymerase 2 was the response to ATP, which not only stimulated processivity by more than 2-fold, but also produced a distinctive pattern in which the enzyme seemed to pause every 10 nucleotides, reaching a value of 40 to 50 nucleotides incorporated. This pattern was observed in some, but not ali, heparin-Sepharose fractions with enzyme activity, suggesting the possibility of different DNA polymerase 2 complexes.

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DNA polymerases from plant cells

Cited by 22 publications

References 58 publications

Plant Enzymes but Not Agrobacterium VirD2 Mediate T-DNA Ligation In Vitro

Plant Enzymes but Not Agrobacterium VirD2 Mediate T-DNA Ligation In Vitro

Gene for proliferating-cell nuclear antigen (DNA polymerase delta auxiliary protein) is present in both mammalian and higher plant genomes.

Studies on the Processivity of Maize DNA Polymerase 2, an [alpha]-Type Enzyme

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