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| The EMBO Journal
(1999) 18, 3834–3844, doi:10.1093/emboj/18.13.3834 |
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| Post-replicative base excision repair in replication foci |
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| Marit Otterlei1, Emma Warbrick2, Toril A. Nagelhus1, 3, Terje Haug1, Geir Slupphaug1, Mansour Akbari1, Per Arne Aas1, Kristin Steinsbekk1, Oddmund Bakke4 and Hans E. Krokan1 |
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1 Institute of Cancer Research and Molecular Biology, Faculty of Medicine, Norwegian University of Science and Technology, N-7005 Trondheim, Norway 2 Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, UK 3 Department of Physics, Norwegian University of Science and Technology, N-7034 Trondheim, Norway 4 Institute of Molecular Cell Biology, Faculty of Biology, University of Oslo, N-0316 Oslo, Norway Received 6 April 1999; Revised 5 May 1999; Accepted 7 May 1999. |
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| Abstract |
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| Base excision repair (BER) is initiated by a DNA glycosylase and is completed by alternative routes, one of which requires proliferating cell nuclear antigen (PCNA) and other proteins also involved in DNA replication. We report that the major nuclear uraci-DNA glycosylase (UNG2) increases in S phase, during which it co-localizes with incorporated BrdUrd in replication foci. Uracil is rapidly removed from replicatively incorporated dUMP residues in isolated nuclei. Neutralizing antibodies to UNG2 inhibit this removal, indicating that UNG2 is the major uraci-DNA glycosylase responsible. PCNA and replication protein A (RPA) co-localize with UNG2 in replication foci, and a direct molecular interaction of UNG2 with PCNA (one binding site) and RPA (two binding sites) was demonstrated using two-hybrid assays, a peptide SPOT assay and enzyme-linked immunosorbent assays. These results demonstrate rapid post-replicative removal of incorporated uracil by UNG2 and indicate the formation of a BER complex that contains UNG2, RPA and PCNA close to the replication fork. |
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| Keywords: proliferating cell nuclear antigen, replication foci, replication protein A, uraci-DNA glycosylase |
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