Mechanism of adenomatous polyposis coli (APC)-mediated blockage of long-patch base excision repair.
Recently, we found an interaction between adenomatous polyposis coli (APC) and DNA polymerase beta (pol-beta) and showed that APC blocks strand-displacement synthesis of long-patch base excision repair (LP-BER) (Narayan, S., Jaiswal, A. S., and Balusu, R. (2005) J. Biol. Chem. 280, 6942-6949); however, the mechanism is not clear. Using an in vivo LP-BER assay system, we now show that the LP-BER is higher in APC-/- cells than in APC+/+ cells. In addition to pol-beta, the pull-down experiments showed that the full-length APC also interacted with flap endonuclease 1 (Fen-1). To further characterize the interaction of APC with pol-beta and Fen-1, we performed a domain-mapping of APC and found that both pol-beta and Fen-1 interact with a 138-amino acids peptide from the APC at the DRI-domain. Our functional assays showed that APC blocks pol-beta-mediated 1-nucleotide (1-nt) as well as strand-displacement synthesis of reduced abasic, nicked-, or 1-nt gapped-DNA substrates. Further studies demonstrated that APC blocks 5'-flap endonuclease as well as the 5'-3' exonuclease activity of Fen-1 resulting in the blockage of LP-BER. From these results, we concluded that APC can have three different effects on the LP-BER pathway. First, APC can block pol-beta-mediated 1-nt incorporation and strand-displacement synthesis. Second, APC can block LP-BER by blocking the coordinated formation and removal of the strand-displaced flap. Third, APC can block LP-BER by blocking hit-and-run synthesis. These studies will have important implications for APC in DNA damage-induced carcinogenesis and chemoprevention.
Jaiswal
Aruna S
AS
Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, Florida 32610, USA.
Balusu
Ramesh
R
Armas
Melissa L
ML
Kundu
Chanakya N
CN
Narayan
Satya
S
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D019951:DNA Polymerase beta / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D004273:DNA, Neoplasm / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D005092:Exonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism; D045585:Flap Endonucleases / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000502:physiology*; D017491:Genes, APC / Q000502:physiology*; D045325:HCT116 Cells; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000235:genetics
D004273:DNA, Neoplasm; D019951:DNA Polymerase beta; D005092:Exonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human