DOI: 10.1073/pnas.1330328100. Suppressed catalytic activity of base excision repair enzymes on rotationally positioned uracil in nucleosomes

Suppressed catalytic activity of base excision repair enzymes on rotationally positioned uracil in nucleosomes

10.1073/pnas.1330328100

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

The majority of DNA in eukaryotic cells exists in the highly condensed structural hierarchy of chromatin, which presents a challenge to DNA repair enzymes in that recognition, incision, and restoration of the original sequence at most sites must take place within these structural constraints. To test base excision repair (BER) activities on chromatin substrates, an in vitro system was developed that uses human uracil DNA glycosylase (UDG), apyrimidinic/apurinic endonuclease (APE), and DNA polymerase β (pol β) on homogeneously damaged, rotationally positioned DNA in nucleosomes. We find that UDG and APE carry out their combined catalytic activities with reduced efficiency on nucleosome substrates (≈10% of that on naked DNA). Furthermore, these enzymes distinguish between two different rotational settings of the lesion on the histone surface, showing a 2- to 3-fold difference in activity between uracil facing ``toward'' and ``away from'' the histones. However, UDG and APE will digest such substrates to completion in a concentration-dependent manner. Conversely, the synthesis activity of pol β is inhibited completely by nucleosome substrates and is independent of enzyme concentration. These results suggest that the first two steps of BER, UDG and APE, may occur ``unassisted'' in chromatin, whereas downstream factors in this pathway (i.e., pol β) may require nucleosome remodeling for efficient DNA BER in at least some regions of chromatin in eukaryotic cells.

Bibliography

Beard, B. C., Wilson, S. H., & Smerdon, M. J. (2003). Suppressed catalytic activity of base excision repair enzymes on rotationally positioned uracil in nucleosomes. Proceedings of the National Academy of Sciences, 100(13), 7465â7470.

Authors 3

Brian C. Beard (first)
Samuel H. Wilson (additional)
Michael J. Smerdon (additional)

References 61 Referenced 134

10.1038/35077232
10.1126/science.1056154
10.1016/S0027-5107(98)00084-0
10.1038/38444
Wolffe A. P. (1998) Chromatin: Structure and Function (Academic San Diego). (10.1016/S1067-5701(98)80005-3)
van Holde K. E. (1988) Chromatin (Springer New York). (10.1007/978-1-4612-3490-6)
10.1093/emboj/18.13.3712
Li, Q. & Wrange, O. (1995) Mol. Cell. Biol. 8, 4375–4384. / Mol. Cell. Biol. (1995)
10.1101/gad.7.12a.2471
10.1021/bi990297h
10.1126/science.1063127
10.1146/annurev.biochem.71.110601.135400
10.1073/pnas.90.17.7915
10.1038/362709a0
10.1016/S0027-5107(02)00258-0
10.1021/bi00077a005
10.1126/science.1948068
10.1021/bi00065a003
10.1126/science.7839142
10.1073/pnas.94.6.2215
Friedberg E. C. Walker G. C. & Siede W. (1995) DNA Repair and Mutagenesis (Am. Soc. Microbiol. Washington DC).
10.1074/jbc.273.33.21203
10.1016/S0022-2836(05)80061-0
10.1093/nar/8.16.3517
10.1093/nar/6.4.1387
10.1093/nar/24.21.4349
10.1093/nar/23.22.4557
10.1074/jbc.274.18.12201
10.1093/nar/29.18.3857
10.1126/science.7624801
10.1074/jbc.271.30.17811
10.1016/S1097-2765(02)00736-0
10.1093/emboj/19.20.5492
10.1021/bi00294a050
10.1073/pnas.79.19.5896
10.1038/35000249
10.1038/384087a0
10.1021/bi9703812
10.1016/S0969-2126(02)00930-9
10.1128/MCB.19.10.6532
10.1093/embo-reports/kvf075
10.1046/j.1432-1033.2002.02888.x
Harrigan J. A. Opresko P. L. Von Kobbe C. Kedar P. S. Prasad R. Wilson S. H. & Bohr V. A. (March 27 2003) J. Biol. Chem. 10.1074/jbc.M213103200.
10.1002/j.1460-2075.1996.tb01056.x
10.1093/nar/28.10.2049
10.1093/emboj/20.22.6530
10.1021/bi962710g
10.1016/S1383-5742(02)00009-1
10.1128/MCB.18.6.3563
10.1006/scel.1995.0031
10.1128/MCB.22.19.6779-6787.2002
10.1093/emboj/20.8.2004
Gaillard H. Fitzgerald D. J. Smith C. L. Peterson C. L. Richmond T. J. & Thoma F. (March 11 2003) J. Biol. Chem. 10.1074/jbc.M300770200.
10.1093/emboj/cdf581
10.1021/bi991771m
10.1016/0022-2836(91)90384-I
10.1073/pnas.87.15.5724
10.1073/pnas.93.4.1370
10.1006/jmbi.1998.2087
10.1128/MCB.22.20.7147-7157.2002
10.1006/jmbi.1997.0916

Dates

Type	When
Created	22 years, 2 months ago (June 24, 2003, 1:47 p.m.)
Deposited	3 years, 4 months ago (April 12, 2022, 11:12 p.m.)
Indexed	3 days, 4 hours ago (Aug. 30, 2025, 12:48 p.m.)
Issued	22 years, 2 months ago (June 10, 2003)
Published	22 years, 2 months ago (June 10, 2003)
Published Online	22 years, 2 months ago (June 10, 2003)
Published Print	22 years, 2 months ago (June 24, 2003)

Funders 0

None

BibTeX

@article{Beard_2003, title={Suppressed catalytic activity of base excision repair enzymes on rotationally positioned uracil in nucleosomes}, volume={100}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.1330328100}, DOI={10.1073/pnas.1330328100}, number={13}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Beard, Brian C. and Wilson, Samuel H. and Smerdon, Michael J.}, year={2003}, month=jun, pages={7465–7470} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        8,
        30
      ]
    ],
    "date-time": "2025-08-30T16:48:06Z",
    "timestamp": 1756572486856
  },
  "reference-count": 61,
  "publisher": "Proceedings of the National Academy of Sciences",
  "issue": "13",
  "content-domain": {
    "domain": [
      "www.pnas.org"
    ],
    "crossmark-restriction": true
  },
  "published-print": {
    "date-parts": [
      [
        2003,
        6,
        24
      ]
    ]
  },
  "abstract": "<jats:p>\n            The majority of DNA in eukaryotic cells exists in the highly condensed  structural hierarchy of chromatin, which presents a challenge to DNA repair  enzymes in that recognition, incision, and restoration of the original  sequence at most sites must take place within these structural constraints. To  test base excision repair (BER) activities on chromatin substrates, an\n            <jats:italic>in  vitro</jats:italic>\n            system was developed that uses human uracil DNA glycosylase (UDG),  apyrimidinic/apurinic endonuclease (APE), and DNA polymerase \u03b2 (pol  \u03b2) on homogeneously damaged, rotationally positioned DNA in nucleosomes.  We find that UDG and APE carry out their combined catalytic activities with  reduced efficiency on nucleosome substrates (\u224810% of that on naked DNA).  Furthermore, these enzymes distinguish between two different rotational  settings of the lesion on the histone surface, showing a 2- to 3-fold  difference in activity between uracil facing ``toward'' and ``away from'' the  histones. However, UDG and APE will digest such substrates to completion in a  concentration-dependent manner. Conversely, the synthesis activity of pol  \u03b2 is inhibited completely by nucleosome substrates and is independent of  enzyme concentration. These results suggest that the first two steps of BER,  UDG and APE, may occur ``unassisted'' in chromatin, whereas downstream factors  in this pathway (i.e., pol \u03b2) may require nucleosome remodeling for  efficient DNA BER in at least some regions of chromatin in eukaryotic  cells.\n          </jats:p>",
  "DOI": "10.1073/pnas.1330328100",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2003,
        6,
        24
      ]
    ],
    "date-time": "2003-06-24T17:47:39Z",
    "timestamp": 1056476859000
  },
  "page": "7465-7470",
  "update-policy": "http://dx.doi.org/10.1073/pnas.cm10313",
  "source": "Crossref",
  "is-referenced-by-count": 134,
  "title": "Suppressed catalytic activity of base excision repair enzymes on  rotationally positioned uracil in nucleosomes",
  "prefix": "10.1073",
  "volume": "100",
  "author": [
    {
      "given": "Brian C.",
      "family": "Beard",
      "sequence": "first",
      "affiliation": [
        {
          "name": "Department of Biochemistry and  Biophysics, School of Molecular Biosciences, Washington State University,  Pullman, WA 99164-4660; and Laboratory of  Structural Biology, National Institute of Environmental Health Sciences,  Research Triangle Park, NC 27709"
        }
      ]
    },
    {
      "given": "Samuel H.",
      "family": "Wilson",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Department of Biochemistry and  Biophysics, School of Molecular Biosciences, Washington State University,  Pullman, WA 99164-4660; and Laboratory of  Structural Biology, National Institute of Environmental Health Sciences,  Research Triangle Park, NC 27709"
        }
      ]
    },
    {
      "given": "Michael J.",
      "family": "Smerdon",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Department of Biochemistry and  Biophysics, School of Molecular Biosciences, Washington State University,  Pullman, WA 99164-4660; and Laboratory of  Structural Biology, National Institute of Environmental Health Sciences,  Research Triangle Park, NC 27709"
        }
      ]
    }
  ],
  "member": "341",
  "published-online": {
    "date-parts": [
      [
        2003,
        6,
        10
      ]
    ]
  },
  "reference": [
    {
      "key": "e_1_3_2_1_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/35077232"
    },
    {
      "key": "e_1_3_2_2_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.1056154"
    },
    {
      "key": "e_1_3_2_3_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0027-5107(98)00084-0"
    },
    {
      "key": "e_1_3_2_4_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/38444"
    },
    {
      "key": "e_1_3_2_5_2",
      "doi-asserted-by": "crossref",
      "unstructured": "Wolffe A. P. (1998) Chromatin: Structure  and Function (Academic San Diego).",
      "DOI": "10.1016/S1067-5701(98)80005-3"
    },
    {
      "key": "e_1_3_2_6_2",
      "doi-asserted-by": "crossref",
      "unstructured": "van Holde K. E. (1988) Chromatin (Springer New York).",
      "DOI": "10.1007/978-1-4612-3490-6"
    },
    {
      "key": "e_1_3_2_7_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/emboj/18.13.3712"
    },
    {
      "key": "e_1_3_2_8_2",
      "first-page": "4375",
      "volume": "8",
      "year": "1995",
      "unstructured": "Li, Q. & Wrange, O. (1995) Mol. Cell. Biol. 8, 4375\u20134384.",
      "journal-title": "Mol. Cell. Biol."
    },
    {
      "key": "e_1_3_2_9_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1101/gad.7.12a.2471"
    },
    {
      "key": "e_1_3_2_10_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi990297h"
    },
    {
      "key": "e_1_3_2_11_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.1063127"
    },
    {
      "key": "e_1_3_2_12_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1146/annurev.biochem.71.110601.135400"
    },
    {
      "key": "e_1_3_2_13_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.90.17.7915"
    },
    {
      "key": "e_1_3_2_14_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/362709a0"
    },
    {
      "key": "e_1_3_2_15_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0027-5107(02)00258-0"
    },
    {
      "key": "e_1_3_2_16_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00077a005"
    },
    {
      "key": "e_1_3_2_17_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.1948068"
    },
    {
      "key": "e_1_3_2_18_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00065a003"
    },
    {
      "key": "e_1_3_2_19_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.7839142"
    },
    {
      "key": "e_1_3_2_20_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.94.6.2215"
    },
    {
      "key": "e_1_3_2_21_2",
      "unstructured": "Friedberg E. C. Walker G. C. & Siede W. (1995) DNA Repair and Mutagenesis (Am. Soc. Microbiol. Washington DC)."
    },
    {
      "key": "e_1_3_2_22_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1074/jbc.273.33.21203"
    },
    {
      "key": "e_1_3_2_23_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0022-2836(05)80061-0"
    },
    {
      "key": "e_1_3_2_24_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/8.16.3517"
    },
    {
      "key": "e_1_3_2_25_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/6.4.1387"
    },
    {
      "key": "e_1_3_2_26_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/24.21.4349"
    },
    {
      "key": "e_1_3_2_27_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/23.22.4557"
    },
    {
      "key": "e_1_3_2_28_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1074/jbc.274.18.12201"
    },
    {
      "key": "e_1_3_2_29_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/29.18.3857"
    },
    {
      "key": "e_1_3_2_30_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.7624801"
    },
    {
      "key": "e_1_3_2_31_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1074/jbc.271.30.17811"
    },
    {
      "key": "e_1_3_2_32_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S1097-2765(02)00736-0"
    },
    {
      "key": "e_1_3_2_33_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/emboj/19.20.5492"
    },
    {
      "key": "e_1_3_2_34_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00294a050"
    },
    {
      "key": "e_1_3_2_35_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.79.19.5896"
    },
    {
      "key": "e_1_3_2_36_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/35000249"
    },
    {
      "key": "e_1_3_2_37_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/384087a0"
    },
    {
      "key": "e_1_3_2_38_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi9703812"
    },
    {
      "key": "e_1_3_2_39_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0969-2126(02)00930-9"
    },
    {
      "key": "e_1_3_2_40_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/MCB.19.10.6532"
    },
    {
      "key": "e_1_3_2_41_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/embo-reports/kvf075"
    },
    {
      "key": "e_1_3_2_42_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1046/j.1432-1033.2002.02888.x"
    },
    {
      "key": "e_1_3_2_43_2",
      "unstructured": "Harrigan J. A. Opresko P. L. Von Kobbe C. Kedar P. S. Prasad R. Wilson S. H. & Bohr V. A. (March 27 2003) J. Biol. Chem. 10.1074/jbc.M213103200."
    },
    {
      "key": "e_1_3_2_44_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/j.1460-2075.1996.tb01056.x"
    },
    {
      "key": "e_1_3_2_45_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/28.10.2049"
    },
    {
      "key": "e_1_3_2_46_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/emboj/20.22.6530"
    },
    {
      "key": "e_1_3_2_47_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi962710g"
    },
    {
      "key": "e_1_3_2_48_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S1383-5742(02)00009-1"
    },
    {
      "key": "e_1_3_2_49_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/MCB.18.6.3563"
    },
    {
      "key": "e_1_3_2_50_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1006/scel.1995.0031"
    },
    {
      "key": "e_1_3_2_51_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/MCB.22.19.6779-6787.2002"
    },
    {
      "key": "e_1_3_2_52_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/emboj/20.8.2004"
    },
    {
      "key": "e_1_3_2_53_2",
      "unstructured": "Gaillard H. Fitzgerald D. J. Smith C. L. Peterson C. L. Richmond T. J. & Thoma F. (March 11 2003) J.  Biol. Chem. 10.1074/jbc.M300770200."
    },
    {
      "key": "e_1_3_2_54_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/emboj/cdf581"
    },
    {
      "key": "e_1_3_2_55_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi991771m"
    },
    {
      "key": "e_1_3_2_56_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0022-2836(91)90384-I"
    },
    {
      "key": "e_1_3_2_57_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.87.15.5724"
    },
    {
      "key": "e_1_3_2_58_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.93.4.1370"
    },
    {
      "key": "e_1_3_2_59_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1006/jmbi.1998.2087"
    },
    {
      "key": "e_1_3_2_60_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/MCB.22.20.7147-7157.2002"
    },
    {
      "key": "e_1_3_2_61_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1006/jmbi.1997.0916"
    }
  ],
  "container-title": "Proceedings of the National Academy of Sciences",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://pnas.org/doi/pdf/10.1073/pnas.1330328100",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2022,
        4,
        13
      ]
    ],
    "date-time": "2022-04-13T03:12:56Z",
    "timestamp": 1649819576000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://pnas.org/doi/full/10.1073/pnas.1330328100"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        2003,
        6,
        10
      ]
    ]
  },
  "references-count": 61,
  "journal-issue": {
    "issue": "13",
    "published-print": {
      "date-parts": [
        [
          2003,
          6,
          24
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1073/pnas.1330328100"
  ],
  "URL": "http://dx.doi.org/10.1073/pnas.1330328100",
  "relation": {},
  "ISSN": [
    "0027-8424",
    "1091-6490"
  ],
  "subject": [],
  "container-title-short": "Proc. Natl. Acad. Sci. U.S.A.",
  "published": {
    "date-parts": [
      [
        2003,
        6,
        10
      ]
    ]
  },
  "assertion": [
    {
      "value": "2003-01-18",
      "order": 0,
      "name": "received",
      "label": "Received",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    },
    {
      "value": "2003-06-10",
      "order": 2,
      "name": "published",
      "label": "Published",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    }
  ]
}