DOI: 10.1111/j.1432-1033.1983.tb07670.x. Reactions <i>in vitro</i> of the DNA polymerase‐primase from <i>Xenopus laevis</i> eggs

Reactions in vitro of the DNA polymerase‐primase from Xenopus laevis eggs

10.1111/j.1432-1033.1983.tb07670.x

Crossref journal-article

Wiley

European Journal of Biochemistry (311)

Abstract

A form of DNA polymerase α was purified several thousandfold from a protein extract of Xenopus laevis eggs. The enzyme effectively converts, in the presence of ribonucleoside triphosphates, a circular single‐stranded phage fd DNA template into a double‐stranded DNA form and, therefore, must be associated with a DNA primase. We first show by gel electrophoresis in the presence of sodium dodecyl sulfate that both enzymatic activities, DNA polymerase and primase, most probably reside on a < 100000‐Da subunit of the DNA polymerase holoenzyme. We then assayed the polymerase‐primase at various template/enzyme ratios and found that the DNA complementary strand sections synthesized in vitro belong to defined size classes in the range of 600–2000 nucleotides, suggesting preferred start and/or stop sites on the fd DNA template strand. We show that the stop sites coincide with stable hairpin structures in fd DNA. We have used a fd DNA template, primed by a restriction fragment of known size, to show that the polymerase‐primase stops at the first stable hairpin structure upstream from the 3′‐OH primer site when the reaction was carried out at 0.1 mM ATP. However, at 2mM ATP the enzyme was able to travers this and other stop sites on the fd DNA template strand leading to the synthesis of 2–4 times longer DNA strands. Our results suggest a role for ATP in the polymerase‐primase‐catalyzed chain‐elongation reaction.

Bibliography

KÃNIG, H., RIEDEL, H. D., & KNIPPERS, R. (1983). Reactions in vitro of the DNA polymeraseâprimase from Xenopus laevis eggs. European Journal of Biochemistry, 135(3), 435â442. Portico.

Authors 3

Herbert KÖNIG (first)
Hans Dieter RIEDEL (additional)
Rolf KNIPPERS (additional)

References 36 Referenced 26

{'key': 'e_1_2_1_2_2', 'first-page': '25', 'volume': '4', 'author': 'Gurdon J. B.', 'year': '1968', 'journal-title': 'Essays in Biochemistry'} / Essays in Biochemistry by Gurdon J. B. (1968)
10.1007/978-1-4684-9933-9_4
10.1016/0092-8674(82)90272-0
10.1016/0092-8674(82)90273-2
10.1016/0092-8674(80)90439-0
10.1093/nar/9.15.3793
10.1016/0012-1606(75)90138-4
10.1016/0092-8674(78)90199-X
10.1146/annurev.bi.49.070180.003211
{'key': 'e_1_2_1_11_2', 'volume-title': 'Enzymes of DNA Synthesis and Processing', 'author': 'Fry M.', 'year': '1982'} / Enzymes of DNA Synthesis and Processing by Fry M. (1982)
10.1093/nar/10.18.5621
10.1016/0092-8674(82)90015-0
10.1073/pnas.79.23.7209
10.1073/pnas.79.8.2523
10.1093/oxfordjournals.jbchem.a133732
10.1146/annurev.bi.49.070180.002225
10.1073/pnas.79.15.4585
10.1073/pnas.78.11.6771
10.1016/S0021-9258(19)86001-5 / J. Biol. Chem. by Mechali M. (1980)
10.1016/S0021-9258(18)93954-2
10.1016/0022-2836(66)90099-4
10.1016/0092-8674(81)90426-8
10.1093/nar/9.8.1825
10.1111/j.1432-1033.1976.tb10152.x
10.1002/j.1460-2075.1983.tb01394.x / EMBO J. by Huebscher U. (1983)
10.1016/S0021-9258(19)83600-1 / J. Biol. Chem. by Banks G. R. (1979)
10.1073/pnas.76.12.6081
10.1093/nar/6.10.3341
10.1073/pnas.78.8.4723
10.1073/pnas.79.6.1791
10.1073/pnas.79.4.983
10.1093/nar/5.12.4495
10.1016/0014-5793(82)81067-3
10.1111/j.1432-1033.1974.tb03364.x
10.1093/nar/10.14.4181
10.1111/j.1432-1033.1979.tb13232.x

Dates

Type	When
Created	20 years, 6 months ago (March 4, 2005, 10:10 a.m.)
Deposited	1 year, 9 months ago (Nov. 23, 2023, 6:09 a.m.)
Indexed	1 day, 18 hours ago (Sept. 4, 2025, 10:13 a.m.)
Issued	41 years, 11 months ago (Oct. 1, 1983)
Published	41 years, 11 months ago (Oct. 1, 1983)
Published Online	20 years, 6 months ago (March 4, 2005)
Published Print	41 years, 11 months ago (Oct. 1, 1983)

Funders 0

None

BibTeX

@article{K_NIG_1983, title={Reactions in vitro of the DNA polymerase‐primase from Xenopus laevis eggs: A role for ATP in chain elongation}, volume={135}, ISSN={1432-1033}, url={http://dx.doi.org/10.1111/j.1432-1033.1983.tb07670.x}, DOI={10.1111/j.1432-1033.1983.tb07670.x}, number={3}, journal={European Journal of Biochemistry}, publisher={Wiley}, author={KÖNIG, Herbert and RIEDEL, Hans Dieter and KNIPPERS, Rolf}, year={1983}, month=oct, pages={435–442} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        9,
        4
      ]
    ],
    "date-time": "2025-09-04T14:13:52Z",
    "timestamp": 1756995232788
  },
  "reference-count": 36,
  "publisher": "Wiley",
  "issue": "3",
  "license": [
    {
      "start": {
        "date-parts": [
          [
            2005,
            3,
            4
          ]
        ],
        "date-time": "2005-03-04T00:00:00Z",
        "timestamp": 1109894400000
      },
      "content-version": "vor",
      "delay-in-days": 7825,
      "URL": "http://onlinelibrary.wiley.com/termsAndConditions#vor"
    }
  ],
  "content-domain": {
    "domain": [],
    "crossmark-restriction": false
  },
  "published-print": {
    "date-parts": [
      [
        1983,
        10
      ]
    ]
  },
  "abstract": "<jats:p>A form of DNA polymerase \u03b1 was purified several thousandfold from a protein extract of <jats:italic>Xenopus laevis</jats:italic> eggs. The enzyme effectively converts, in the presence of ribonucleoside triphosphates, a circular single\u2010stranded phage fd DNA template into a double\u2010stranded DNA form and, therefore, must be associated with a DNA primase. We first show by gel electrophoresis in the presence of sodium dodecyl sulfate that both enzymatic activities, DNA polymerase and primase, most probably reside on a &lt; 100000\u2010Da subunit of the DNA polymerase holoenzyme. We then assayed the polymerase\u2010primase at various template/enzyme ratios and found that the DNA complementary strand sections synthesized <jats:italic>in vitro</jats:italic> belong to defined size classes in the range of 600\u20132000 nucleotides, suggesting preferred start and/or stop sites on the fd DNA template strand. We show that the stop sites coincide with stable hairpin structures in fd DNA. We have used a fd DNA template, primed by a restriction fragment of known size, to show that the polymerase\u2010primase stops at the first stable hairpin structure upstream from the 3\u2032\u2010OH primer site when the reaction was carried out at 0.1 mM ATP. However, at 2mM ATP the enzyme was able to travers this and other stop sites on the fd DNA template strand leading to the synthesis of 2\u20134 times longer DNA strands. Our results suggest a role for ATP in the polymerase\u2010primase\u2010catalyzed chain\u2010elongation reaction.</jats:p>",
  "DOI": "10.1111/j.1432-1033.1983.tb07670.x",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2005,
        3,
        4
      ]
    ],
    "date-time": "2005-03-04T15:10:58Z",
    "timestamp": 1109949058000
  },
  "page": "435-442",
  "source": "Crossref",
  "is-referenced-by-count": 26,
  "title": "Reactions <i>in vitro</i> of the DNA polymerase\u2010primase from <i>Xenopus laevis</i> eggs",
  "prefix": "10.1111",
  "volume": "135",
  "author": [
    {
      "given": "Herbert",
      "family": "K\u00d6NIG",
      "sequence": "first",
      "affiliation": []
    },
    {
      "given": "Hans Dieter",
      "family": "RIEDEL",
      "sequence": "additional",
      "affiliation": []
    },
    {
      "given": "Rolf",
      "family": "KNIPPERS",
      "sequence": "additional",
      "affiliation": []
    }
  ],
  "member": "311",
  "published-online": {
    "date-parts": [
      [
        2005,
        3,
        4
      ]
    ]
  },
  "reference": [
    {
      "key": "e_1_2_1_2_2",
      "first-page": "25",
      "volume": "4",
      "author": "Gurdon J. B.",
      "year": "1968",
      "journal-title": "Essays in Biochemistry"
    },
    {
      "key": "e_1_2_1_3_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1007/978-1-4684-9933-9_4"
    },
    {
      "key": "e_1_2_1_4_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(82)90272-0"
    },
    {
      "key": "e_1_2_1_5_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(82)90273-2"
    },
    {
      "key": "e_1_2_1_6_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(80)90439-0"
    },
    {
      "key": "e_1_2_1_7_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/9.15.3793"
    },
    {
      "key": "e_1_2_1_8_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0012-1606(75)90138-4"
    },
    {
      "key": "e_1_2_1_9_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(78)90199-X"
    },
    {
      "key": "e_1_2_1_10_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1146/annurev.bi.49.070180.003211"
    },
    {
      "key": "e_1_2_1_11_2",
      "volume-title": "Enzymes of DNA Synthesis and Processing",
      "author": "Fry M.",
      "year": "1982"
    },
    {
      "key": "e_1_2_1_12_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/10.18.5621"
    },
    {
      "key": "e_1_2_1_13_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(82)90015-0"
    },
    {
      "key": "e_1_2_1_14_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.79.23.7209"
    },
    {
      "key": "e_1_2_1_15_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.79.8.2523"
    },
    {
      "key": "e_1_2_1_16_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/oxfordjournals.jbchem.a133732"
    },
    {
      "key": "e_1_2_1_17_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1146/annurev.bi.49.070180.002225"
    },
    {
      "key": "e_1_2_1_18_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.79.15.4585"
    },
    {
      "key": "e_1_2_1_19_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.78.11.6771"
    },
    {
      "key": "e_1_2_1_20_2",
      "doi-asserted-by": "crossref",
      "first-page": "2114",
      "DOI": "10.1016/S0021-9258(19)86001-5",
      "volume": "255",
      "author": "Mechali M.",
      "year": "1980",
      "journal-title": "J. Biol. Chem."
    },
    {
      "key": "e_1_2_1_21_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9258(18)93954-2"
    },
    {
      "key": "e_1_2_1_22_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0022-2836(66)90099-4"
    },
    {
      "key": "e_1_2_1_23_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(81)90426-8"
    },
    {
      "key": "e_1_2_1_24_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/9.8.1825"
    },
    {
      "key": "e_1_2_1_25_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1432-1033.1976.tb10152.x"
    },
    {
      "key": "e_1_2_1_26_2",
      "doi-asserted-by": "crossref",
      "first-page": "133",
      "DOI": "10.1002/j.1460-2075.1983.tb01394.x",
      "volume": "2",
      "author": "Huebscher U.",
      "year": "1983",
      "journal-title": "EMBO J."
    },
    {
      "key": "e_1_2_1_27_2",
      "doi-asserted-by": "crossref",
      "first-page": "9886",
      "DOI": "10.1016/S0021-9258(19)83600-1",
      "volume": "254",
      "author": "Banks G. R.",
      "year": "1979",
      "journal-title": "J. Biol. Chem."
    },
    {
      "key": "e_1_2_1_28_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.76.12.6081"
    },
    {
      "key": "e_1_2_1_29_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/6.10.3341"
    },
    {
      "key": "e_1_2_1_30_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.78.8.4723"
    },
    {
      "key": "e_1_2_1_31_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.79.6.1791"
    },
    {
      "key": "e_1_2_1_32_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.79.4.983"
    },
    {
      "key": "e_1_2_1_33_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/5.12.4495"
    },
    {
      "key": "e_1_2_1_34_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0014-5793(82)81067-3"
    },
    {
      "key": "e_1_2_1_35_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1432-1033.1974.tb03364.x"
    },
    {
      "key": "e_1_2_1_36_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/10.14.4181"
    },
    {
      "key": "e_1_2_1_37_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1432-1033.1979.tb13232.x"
    }
  ],
  "container-title": "European Journal of Biochemistry",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1111%2Fj.1432-1033.1983.tb07670.x",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "text-mining"
    },
    {
      "URL": "https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1983.tb07670.x",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2023,
        11,
        23
      ]
    ],
    "date-time": "2023-11-23T11:09:42Z",
    "timestamp": 1700737782000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1983.tb07670.x"
    }
  },
  "subtitle": [
    "A role for ATP in chain elongation"
  ],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        1983,
        10
      ]
    ]
  },
  "references-count": 36,
  "journal-issue": {
    "issue": "3",
    "published-print": {
      "date-parts": [
        [
          1983,
          10
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1111/j.1432-1033.1983.tb07670.x"
  ],
  "URL": "http://dx.doi.org/10.1111/j.1432-1033.1983.tb07670.x",
  "relation": {},
  "ISSN": [
    "0014-2956",
    "1432-1033"
  ],
  "subject": [],
  "container-title-short": "European Journal of Biochemistry",
  "published": {
    "date-parts": [
      [
        1983,
        10
      ]
    ]
  }
}