Partial purification and characterization of a DNA helicase from chloroplasts of Glycine max

Cannon, Gordon C.; Heinhorst, Sabine

doi:10.1007/bf00019162

Cited by 17 publications

(8 citation statements)

References 21 publications

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“…The pea chloroplast DNA helicase moves in the 3'-to-5' direction along the bound strand similar to the previously described human DNA helicases I, 11, 111, V and VI [13][14][15][16][17][18][19][20][21][22][23][24][25]17, 181 and simian virus (SV)-40 T antigen [30]. Most prokaryotic DNA helicases that are involved in origin activation and growing-fork movement move in the 5'40-3' direction with respect to the strand to which they are bound.…”

Section: Discussionmentioning

confidence: 87%

“…The DNA substrate used to measure the unwinding activity during purification and most of the characterization of the helicase consisted of a '*Plabeled 47-base oligonucleotide annealed through its central 17 nucleotides (positions [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] to M13mp19 ss DNA. This duplex contained 5' and 3' overhanging tails.…”

Section: Resultsmentioning

confidence: 99%

“…The specific direction of unwinding by DNA helicases is still unknown. The second report from plant was of a DNA helicase from chloroplast of Glycine max [20]. Neither of these helicases were well characterized or purified to near homogeneity.…”

Section: Discussionmentioning

confidence: 99%

“…Six DNA helicases have been isolated from human cells by Tuteja et al . In plants, there are two reports of DNA helicases, one in lily [19] and the other in soybean [20]. In both of these studies, the enzymes were not purified to homogeneity or extensively characterized.…”

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confidence: 99%

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Purification and Characterization of a DNA Helicase from Pea Chloroplast that Translocates in the 3′‐to‐5′ Direction

Tuteja

Phan

Tewari

1996

European Journal of Biochemistry

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An ATP-dependent DNA helicase has been purified to near homogeneity from pea chloroplasts. The enzyme is a homodimer of 68-kDa subunits. The purified enzyme shows DNA-dependent ATPase activity and is devoid of DNA polymerase, DNA topoisomerase, DNA ligase or nuclease activities. The enzyme requires Mg2' or MnZ+ for its maximum activity. ATP is the most favoured cofactor for this enzyme while other NTP or dNTP are poorly utilized. Pea chloroplast DNA helicase can unwind a 17-bp duplex whether it has unpaired single-stranded tails at both the 5' end and 3' end, at the 5' end or at the 3' end only, or at neither end. However, it fails to act on a blunt-ended 17-bp duplex DNA. The enzyme moves unidirectionally from 3' to 5' along the bound strand. The unwinding activity is inhibited by the intercalating drugs nogalamycin and daunorubicine.Keywords: unwinding enzyme ; DNA helicase ; DNA-dependent adenosine triphosphatase ; chloroplast ; replication.The mechanism of DNA replication has been well defined in plasmids, bacteriophages, bacteria, viruses and, to a lesser extent, in yeast [1]. Most studies of the replication of DNA in plants have investigated chloroplast DNA [2]. The replicative intermediates of chloroplast DNA molecules of 120-160 kbp from higher plants are well characterized and the origins of replication have been mapped for pea chloroplast DNA and Chlumydonzonus chloroplast DNA [3-61. Of the enzymes involved in DNA replication, only DNA polymerase [7] and DNA topoisomerase [8, 91 have been purified from chloroplasts. The presence of DNA primase has also been reported in pea chloroplasts (101. DNA helicases are an important class of enzymes that are involved in replication, recombination and repair of DNA [I, 11, 121. These enzymes unwind duplex DNA and provide single-stranded DNA template. Many DNA helicases have been isolated from bacteriophage, bacterial, viral and eukaryotic systems [ l l , 121. Multiple DNA helicases are present in a cell because of the different structural requirements of the substrates at various stages of repair, replication and recombination. In Escherichia coli functions for most of the DNA helicases have been attributed [ll]. Six DNA helicases have been isolated from human cells by . In plants, there are two reports of DNA helicases, one in lily [19] and the other in soybean [20]. In both of these studies, the enzymes were not purified to homogeneity or extensively characterized.We now report the purification and detailed characterization of a DNA helicase from pea chloroplast, which is a homodimer of 68-kDa subunits and requires divalent cations for activity.

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Section: Discussionmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Purification and Characterization of a DNA Helicase from Pea Chloroplast that Translocates in the 3′‐to‐5′ Direction

Tuteja

Phan

Tewari

1996

European Journal of Biochemistry

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“…Supercoiled plasmid vector (pBR322) D N A lacking any c t D N A insert was a p o o r template for the extract (Table 1). In addition to D N A polymerase activity, the extract also contains D N A t o p o i s o m e r a s e I, gyrase, D N A helicase [5], R N A p o l y m e r a s e activities as well as strong D N A binding activities (details to be presented elsewhere).…”

Section: Purification Of a Soybean Chloroplast Extract With In Vitro mentioning

confidence: 99%

Analysis of soybean chloroplast DNA replication by two-dimensional gel electrophoresis

et al. 1993

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Chloroplast DNA replication was studied in the green, autotrophic suspension culture line SB-1 of Glycine max. Three regions (restriction fragments Sac I 14.5, Pvu II 4.1 and Pvu II 14.8) on the plastome were identified that displayed significantly higher template activity in in vitro DNA replication assays than all other cloned restriction fragments of the organelle genome, suggesting that these clones contain sequences that are able to direct initiation of DNA replication in vitro. In order to confirm that the potential in vitro origin sites are functional in vivo as well, replication intermediates were analyzed by two-dimensional gel electrophoresis using cloned restriction fragments as probes. The two Pvu II fragments that supported deoxynucleotide incorporation in vitro apparently do not contain a functional in vivo replication origin since replication intermediates from these areas of the plastome represent only fork structures. The Sac I 14.5 chloroplast DNA fragment, on the other hand, showed intermediates consistent with a replication bubble originating within its borders, which is indicative of an active in vivo origin. Closer examination of cloned Sac I 14.5 sub-fragments confirmed high template activity in vitro for two, S/B 5 and S/B 3, which also seem to contain origin sites utilized in vivo as determined by two-dimensional gel electrophoresis. The types of replication intermediate patterns obtained for these sub-fragments are consistent with the double D-loop model for chloroplast DNA replication with both origins being located in the large unique region of the plastome [17,18]. This is the first report of a chloroplast DNA replication origin in higher plants that has been directly tested for in vivo function.

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DNA replication, recombination, and repair in plastids

Day

Madesis

2007

Topics in Current Genetics

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Partial purification and characterization of a DNA helicase from chloroplasts of Glycine max

Cited by 17 publications

References 21 publications

Purification and Characterization of a DNA Helicase from Pea Chloroplast that Translocates in the 3′‐to‐5′ Direction

Purification and Characterization of a DNA Helicase from Pea Chloroplast that Translocates in the 3′‐to‐5′ Direction

Analysis of soybean chloroplast DNA replication by two-dimensional gel electrophoresis

DNA replication, recombination, and repair in plastids

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