Abstract
Recently, photoaffinity labeling experiments with mouse cell extracts suggested that PARP-1 functions as a surveillance protein for a stalled BER intermediate. To further understand the role of PARP-1 in BER, we examined the DNA synthesis and flap excision steps in long patch BER using a reconstituted system containing a 34-base pair BER substrate and five purified human enzymes: uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta, flap endonuclease-1 (FEN-1), and PARP-1. PARP-1 stimulates strand displacement DNA synthesis by DNA polymerase beta in this system; this stimulation is dependent on the presence of FEN-1. PARP-1 and FEN-1, therefore, cooperate to activate long patch BER. The results are discussed in the context of a model for BER sub-pathway choice, illustrating a dual role for PARP-1 as a surveillance protein for a stalled BER intermediate and an activating factor for long patch BER DNA synthesis.
Highlights
From the ‡Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
Suggest that some of the sequential steps may be coordinated such that the product of one reaction can be handed off to the enzyme in the series [11]. In keeping with this idea, photoaffinity cross-linking studies of Base excision repair (BER) intermediates in crude cell extracts revealed that poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA nick surveillance protein that binds only weakly to BER intermediates when single nucleotide BER proceeds normally to completion
The DNA products, in this system without a DNA ligase, are a 16-nucleotide molecule with [␣-32P]dNMP incorporated at position 16 representing a single nucleotide BER intermediate, plus longer products (Ͼ16-nucleotide-long molecules) representing strand displacement synthesis and long patch BER intermediates
Summary
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol 276, No 35, Issue of August 31, pp. 32411–32414, 2001. Suggest that some of the sequential steps may be coordinated such that the product of one reaction can be handed off to the enzyme in the series [11] In keeping with this idea, photoaffinity cross-linking studies of BER intermediates in crude cell extracts revealed that poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA nick surveillance protein that binds only weakly to BER intermediates when single nucleotide BER proceeds normally to completion. When single nucleotide BER is stalled by a block in the excision step, PARP-1 binds strongly to the BER intermediate, along with apurinic/ apyrimidinic endonuclease (APE), DNA polymerase  (-pol), and flap endonuclease-1 (FEN-1) [12]. Our results show that PARP-1, along with FEN-1, stimulates -pol strand displacement DNA synthesis of the long patch BER excision patch
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