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J Biol Chem. 1999 Nov 19;274(47):33703-8.

Reconstitution of proliferating cell nuclear antigen-dependent repair of apurinic/apyrimidinic sites with purified human proteins.

Author information

1
Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA. y_matsumoto@fccc.edu

Abstract

An apurinic/apyrimidinic (AP) site is one of the most abundant lesions spontaneously generated in living cells and is also a reaction intermediate in base excision repair. In higher eukaryotes, there are two alternative pathways for base excision repair: a DNA polymerase beta-dependent pathway and a proliferating cell nuclear antigen (PCNA)-dependent pathway. Here we have reconstituted PCNA-dependent repair of AP sites with six purified human proteins: AP endonuclease, replication factor C, PCNA, flap endonuclease 1 (FEN1), DNA polymerase delta, and DNA ligase I. The length of nucleotides replaced during the repair reaction (patch size) was predominantly two nucleotides, although longer patches of up to seven nucleotides could be detected. Neither replication protein A nor Ku70/80 enhanced the repair activity in this system. Disruption of the PCNA-binding site of either FEN1 or DNA ligase I significantly reduced efficiency of AP site repair but did not affect repair patch size.

PMID:
10559261
DOI:
10.1074/jbc.274.47.33703
[Indexed for MEDLINE]
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