Abstract
Interactions of APE1 (human apurinic/apyrimidinic endonuclease 1) and DNA polymerase β with various DNA structures imitating intermediates of DNA repair and replication were investigated by gel retardation and photoaffinity labeling. Photoaffinity labeling of APE1 and DNA polymerase β was accomplished by DNA containing photoreactive group at the 3′-end in mouse embryonic fibroblast (MEF) cell extract or for purified proteins. On the whole, modification efficiency was the same for MEF-extract proteins and for purified APE1 and DNA polymerase β depending on the nature of the 5′-group of a nick/gap in the DNA substrate. Some of DNA duplexes used in this work can be considered as short-patch (DNA with the 5′-phosphate group in the nick/gap) or long-patch (DNA containing 5′-sugar phosphate or 5′-flap) base excision repair (BER) intermediates. Other DNA duplexes (3′-recessed DNA and DNA with the 5′-hydroxyl group in the nick/gap) have no relation to intermediates forming in the course of BER. As shown by both methods, APE1 binds with the highest efficiency to DNA substrate containing 5′-sugar phosphate group in the nick/gap, whereas DNA polymerase β binds to DNA duplex with a mononucleotide gap flanked by the 5′-p group. When APE1 and DNA polymerase β are both present, a ternary complex APE1-DNA polymerase β-DNA is formed with the highest efficiency with DNA product of APE1 endonuclease activity and with DNA containing 5′-flap or mononucleotide-gapped DNA with 5′-p group. It was found that APE1 stimulates DNA synthesis catalyzed by DNA polymerase β, and a human X-ray repair cross-complementing group 1 protein (XRCC1) stimulates APE1 3′–5′ exonuclease activity on 3′-recessed DNA duplex.
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Abbreviations
- AP site:
-
apurinic/apyrimidinic site
- AP-dsDNA:
-
double-strand DNA containing AP site in the middle of one of strands
- AP-ssDNA:
-
single-strand DNA containing AP site in the middle of strand
- APE1:
-
human apurinic/apyrimidinic endonuclease 1
- BER:
-
base excision repair
- β polymerase:
-
DNA polymerase β
- FAPdCTP (FAPdCMP):
-
exo-N-[2-N-(N-(4-azido-2,5-difluoro-3-chloropyridin-6-yl)-3-aminopropionyl)-aminoethyl]-2′-deoxycytidine-5′-triphosphate (monophosphate)
- MEF extract:
-
mouse embryonic fibroblast cell extract
- tetrahydrofuran or F:
-
3′-hydroxy-2′-hydroxymethyltetrahydrofuran
- 5′-pF:
-
5′-tetrahydrofuran phosphate
- XRCC1:
-
human X-ray repair cross-complementing group 1 protein
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Original Russian Text © N. S. Dyrkheeva, S. N. Khodyreva, O. I. Lavrik, 2008, published in Biokhimiya, 2008, Vol. 73, No. 3, pp. 322–335.
Originally published in Biochemistry (Moscow) On-Line Papers in Press, a Manuscript BM07-259, January 7, 2008.
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Dyrkheeva, N.S., Khodyreva, S.N. & Lavrik, O.I. Interaction of APE1 and other repair proteins with DNA duplexes imitating intermediates of DNA repair and replication. Biochemistry Moscow 73, 261–272 (2008). https://doi.org/10.1134/S0006297908030048
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DOI: https://doi.org/10.1134/S0006297908030048