Post-replicative base excision repair in replication foci.
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 uracil-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 uracil-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.
Institute of Cancer Research and Molecular Biology, Faculty of Medicine, Norwegian University of Science and Technology, N-7005 Trondheim, Norway.
M
M
Otterlei
E
E
Warbrick
T A
TA
Nagelhus
T
T
Haug
G
G
Slupphaug
M
M
Akbari
P A
PA
Aas
K
K
Steinsbekk
O
O
Bakke
H E
HE
Krokan
D000595:Amino Acid Sequence; D020137:Base Pair Mismatch / Q000235:genetics*; D001665:Binding Sites; D002453:Cell Cycle; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D045647:DNA Glycosylases; D004260:DNA Repair / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051716:Replication Protein A; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase; D015003:Yeasts / Q000166:cytology / Q000235:genetics
D004268:DNA-Binding Proteins; D003856:Deoxyuracil Nucleotides; D010446:Peptide Fragments; D018809:Proliferating Cell Nuclear Antigen; C497167:RPA1 protein, human; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A; D014498:Uracil; D004247:DNA; C007267:2'-deoxyuridylic acid; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase