Molecular cloning of the cDNA for the catalytic subunit of plant DNA polymerase α and its cell‐cycle dependent expression

Yokoi, Masayuki; Ito, Masaki; Izumi, Masako; Miyazawa, Hiroshi; Nakai, Hirokazu; Hanaoka, Fumio

doi:10.1046/j.1365-2443.1997.1560354.x

Cited by 18 publications

(14 citation statements)

References 41 publications

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“…3A and coexpressed with p54-p46 in COS-1 cells. An alignment of amino acid sequences of DNA polymerase ␣ from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster, Trypanosoma brucei, humans, mice, and Oryza sativa allowed us to choose domain boundaries for construction of mutants with deletions (22,44). The subcellular distribution of the deletion mutants was determined by immunodetection with an anti-six-His monoclonal antibody.…”

Section: Resultsmentioning

confidence: 99%

“…However, even though the precise roles of DNA polymerases ␣ and ␦ have been established for the SV40 DNA replication system, the way in which these enzymes function during replication of the chromosome is still not clear. Namely, we are ignorant about the architecture of the subunit assemblies in the replication complexes, the way in which the activities of these complexes are regulated, the coordination that must exist between these polymerases at the replication fork, and which DNA polymerase, ␦ or ε, participates in the elongation of the leading strand and lagging strand (3,4,34).DNA polymerases ␣, ␦, ε, and contain amino acid sequences that are conserved among a wide range of DNA polymerases, indicating that these polymerases belong to the class B DNA polymerase family (32,42,44). During this decade, molecular cloning analysis has shown that the large subunits of all these DNA polymerases comprise the catalytic activity, whereas the functions of the smaller subunits, with the exception of the primase subunit, still remain uncertain (12,34,42).…”

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confidence: 99%

“…DNA polymerases ␣, ␦, ε, and contain amino acid sequences that are conserved among a wide range of DNA polymerases, indicating that these polymerases belong to the class B DNA polymerase family (32,42,44). During this decade, molecular cloning analysis has shown that the large subunits of all these DNA polymerases comprise the catalytic activity, whereas the functions of the smaller subunits, with the exception of the primase subunit, still remain uncertain (12,34,42).…”

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confidence: 99%

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Molecular Architecture of the Mouse DNA Polymerase α-Primase Complex

Mizuno

Yamagishi

Miyazawa

et al. 1999

Molecular and Cellular Biology

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The DNA polymerase ␣-primase complex is the only enzyme that provides RNA-DNA primers for chromosomal DNA replication in eukaryotes. Mouse DNA polymerase ␣ has been shown to consist of four subunits, p180, p68, p54, and p46. To characterize the domain structures and subunit requirements for the assembly of the complex, we constructed eukaryotic polycistronic cDNA expression plasmids expressing pairwise the four subunits of DNA polymerase ␣. In addition, the constructs contained an internal ribosome entry site derived from poliovirus. The constructs were transfected in different combinations with vectors expressing single subunits to allow the simultaneous expression of three or four of the subunits in cultured mammalian cells. We demonstrate that the carboxyl-terminal region of p180 (residues 1235 to 1465) is essential for its interaction with both p68 and p54-p46 by immunohistochemical analysis and coprecipitation studies with antibodies. Mutations in the putative zinc fingers present in the carboxyl terminus of p180 abolished the interaction with p68 completely, although the mutants were still capable of interacting with p54-p46. Furthermore, the aminoterminal region (residues 1 to 329) and the carboxyl-terminal region (residues 1280 to 1465) were revealed to be dispensable for DNA polymerase activity. Thus, we can divide the p180 subunit into three domains. The first is the amino-terminal domain (residues 1 to 329), which is dispensable for both polymerase activity and subunit assembly. The second is the minimal core domain (residues 330 to 1279), required for polymerase activity. The third is the carboxyl-terminal domain (residues 1280 to 1465), which is dispensable for polymerase activity but required for the interaction with the other three subunits. Taken together, these results allow us to propose the first structural model for the DNA polymerase ␣-primase complex in terms of subunit assembly, domain structure, and stepwise formation at the cellular level.In mammalian cells, six distinct DNA polymerases, ␣, ␤, ␥, ␦, ε, and , have been cloned so far (3,13,42). Among these, DNA polymerases ␣, ␦, and ε are considered to be involved in chromosomal DNA replication. DNA polymerase ␣ is the only enzyme that is tightly coupled to DNA primase. Therefore, DNA polymerase ␣ has been considered to provide RNA-DNA primers for the initiation of leading-strand synthesis as well as Okazaki fragment synthesis on the lagging strand (12,34,42). By use of the simian virus 40 (SV40) in vitro DNA replication system, it was shown that DNA polymerase ␣ plays a role in the initiation of DNA synthesis by providing RNA-DNA primers for both leading-strand synthesis and laggingstrand synthesis and that DNA polymerase ␦ extensively elongates these primers through a polymerase switch mechanism (40). However, even though the precise roles of DNA polymerases ␣ and ␦ have been established for the SV40 DNA replication system, the way in which these enzymes function during replication of the chromosome is still not clear. Namely, we are ign...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Molecular Architecture of the Mouse DNA Polymerase α-Primase Complex

Mizuno

Yamagishi

Miyazawa

et al. 1999

Molecular and Cellular Biology

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“…Protein complexes containing polymerase and primase activity have been purified from a variety of plant systems (Coello and Vazquez-Ramos, 1995;Garcia et al, 2002), demonstrating that a POLA-like function exists in plants. However, sequence homology has been investigated only for the POLA1 subunit in rice (Yokoi et al, 1997). Rice POLA1 was originally reported to be shorter than other eukaryotic POLA1 homologs, due to a truncated N terminus (Yokoi et al, 1997).…”

Section: Elongation Complexmentioning

confidence: 99%

Genome-Wide Analysis of the Core DNA Replication Machinery in the Higher Plants Arabidopsis and Rice

et al. 2007

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Core DNA replication proteins mediate the initiation, elongation, and Okazaki fragment maturation functions of DNA replication. Although this process is generally conserved in eukaryotes, important differences in the molecular architecture of the DNA replication machine and the function of individual subunits have been reported in various model systems. We have combined genome-wide bioinformatic analyses of Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) with published experimental data to provide a comprehensive view of the core DNA replication machinery in plants. Many components identified in this analysis have not been studied previously in plant systems, including the GINS (go ichi ni san) complex (PSF1, PSF2, PSF3, and SLD5), MCM8, MCM9, MCM10, NOC3, POLA2, POLA3, POLA4, POLD3, POLD4, and RNASEH2. Our results indicate that the core DNA replication machinery from plants is more similar to vertebrates than single-celled yeasts (Saccharomyces cerevisiae), suggesting that animal models may be more relevant to plant systems. However, we also uncovered some important differences between plants and vertebrate machinery. For example, we did not identify geminin or RNASEH1 genes in plants. Our analyses also indicate that plants may be unique among eukaryotes in that they have multiple copies of numerous core DNA replication genes. This finding raises the question of whether specialized functions have evolved in some cases. This analysis establishes that the core DNA replication machinery is highly conserved across plant species and displays many features in common with other eukaryotes and some characteristics that are unique to plants.DNA replication depends on the coordinated action of numerous multiprotein complexes. At the simplest level, it requires an initiator to establish the site of replication initiation, a helicase to unwind DNA, a polymerase to synthesize new DNA, and machinery to process the Okazaki fragments generated during discontinuous synthesis. Much is known about the DNA replication machinery in yeast (Saccharomyces cerevisiae) and animal model systems, but relatively little is known about the apparatus in plants. To gain insight into plant DNA replication components, we have combined published experimental information with our own bioinformatic analysis of genomic sequence data to examine the core DNA replication machinery in the model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa).Figure 1 depicts a model eukaryotic DNA replication fork and illustrates the protein complexes known or suspected to be part of the core DNA replication machine. These complexes mediate the initiation, elongation, and maturation stages of DNA replication and, as such, constitute the core eukaryotic DNA replication machinery. The events leading to the formation of an active DNA replication fork occur in a stepwise fashion, but our understanding of the timing and specific details of how these events unfold in diverse eukaryotes is limited, and there are a growing number of examples of vari...

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“…However, only fragmentary information is available. Apart from basic descriptions of rice (Oryza sativa) genes coding for the homologs of the catalytic subunit of Pola, of two subunits of Pold, and of the replication accessory proteins FEN1 and PCNA (Yokoi et al, 1997;Kimura et al, 2001;Uchiyama et al, 2002), most functional studies of core replication genes, including mutational analysis, were performed in Arabidopsis thaliana. Of the three replicative DNA polymerases, the catalytic subunit of Pola (INCURVATA2) (Barrero et al, 2007) and two of the four subunits of Pol« (Jenik et al, 2005;Ronceret et al, 2005;Yin et al, 2009) were characterized.…”

Section: Introductionmentioning

confidence: 99%

Replication Stress Leads to Genome Instabilities inArabidopsisDNA Polymerase Δ Mutants

et al. 2009

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Impeded DNA replication or a deficiency of its control may critically threaten the genetic information of cells, possibly resulting in genome alterations, such as gross chromosomal translocations, microsatellite instabilities, or increased rates of homologous recombination (HR). We examined an Arabidopsis thaliana line derived from a forward genetic screen, which exhibits an elevated frequency of somatic HR. These HR events originate from replication stress in endoreduplicating cells caused by reduced expression of the gene coding for the catalytic subunit of the DNA polymerase d (POLd1). The analysis of recombination types induced by diverse alleles of pold1 and by replication inhibitors allows the conclusion that two not mutually exclusive mechanisms lead to the generation of recombinogenic breaks at replication forks. In plants with weak pold1 alleles, we observe genome instabilities predominantly at sites with inverted repeats, suggesting the formation and processing of aberrant secondary DNA structures as a result of the accumulation of unreplicated DNA. Stalled and collapsed replication forks account for the more drastic enhancement of HR in plants with strong pold1 mutant alleles. Our data suggest that efficient progression of DNA replication, foremost on the lagging strand, relies on the physiological level of the polymerase d complex and that even a minor disturbance of the replication process critically threatens genomic integrity of Arabidopsis cells.

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Molecular cloning of the cDNA for the catalytic subunit of plant DNA polymerase α and its cell‐cycle dependent expression

Cited by 18 publications

References 41 publications

Molecular Architecture of the Mouse DNA Polymerase α-Primase Complex

Molecular Architecture of the Mouse DNA Polymerase α-Primase Complex

Genome-Wide Analysis of the Core DNA Replication Machinery in the Higher Plants Arabidopsis and Rice

Replication Stress Leads to Genome Instabilities inArabidopsisDNA Polymerase Δ Mutants

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