Structure/function analysis of the interaction of adenomatous polyposis coli with DNA polymerase beta and its implications for base excision repair.
Mutations in the adenomatous polyposis coli (APC) gene are associated with an early onset of colorectal carcinogenesis. Previously, we described a novel role for the APC polypeptide in base excision repair (BER). The single-nucleotide (SN) and long-patch (LP) BER pathways act to repair the abasic sites in DNA that are induced by stressors, such as spontaneous oxidation/reduction, alkylation, and hyperthermia. We have shown that APC interacts with DNA polymerase beta (Pol-beta) and flap endonuclease 1 (Fen-1) and blocks Pol-beta-directed strand-displacement synthesis. In this study, we have mapped the APC interaction site in Pol-beta and have found that Thr79, Lys81, and Arg83 of Pol-beta were critical for its interaction with APC. The Pol-beta protein (T79A/K81A/R83A) blocked strand-displacement DNA synthesis in which tetrahydrofuran was used as DNA substrate. We further showed that the APC-mediated blockage of LP-BER was due to inhibition of Fen-1 activity. Analysis of the APC-mediated blockage of SN-BER indicated that the interaction of APC with Pol-beta blocked SN-BER activity by inhibiting Pol-beta-directed deoxyribose phosphate lyase activity. Collectively, our findings indicate that APC blocked both Pol-beta-directed SN- and LP-BER pathways and increased sensitivity of cells to alkylation induced DNA damage.
Balusu
Ramesh
R
Department of Anatomy and Cell Biology, UF Shands Cancer Center, University of Florida, Gainesville, Florida 32610, USA.
Jaiswal
Aruna S
AS
Armas
Melissa L
ML
Kundu
Chanakya N
CN
Bloom
Linda B
LB
Narayan
Satya
S
D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics*; D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology*; D006801:Humans; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008958:Models, Molecular; D019761:Phosphorus-Oxygen Lyases / Q000378:metabolism
D025601:Adenomatous Polyposis Coli Protein; D008741:Methyl Methanesulfonate; C423906:5'-deoxyribose phosphate lyase; D019951:DNA Polymerase beta; D019761:Phosphorus-Oxygen Lyases