Interaction of APE1 and other repair proteins with DNA duplexes imitating intermediates of DNA repair and replication.
Interactions of APE1 (human apurinic/apyrimidinic endonuclease 1) and DNA polymerase beta 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 beta 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 beta 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 beta binds to DNA duplex with a mononucleotide gap flanked by the 5 -p group. When APE1 and DNA polymerase beta are both present, a ternary complex APE1-DNA polymerase beta-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 beta, and a human X-ray repair cross-complementing group 1 protein (XRCC1) stimulates APE1 3 -5 exonuclease activity on 3 -recessed DNA duplex.
Dyrkheeva
N S
NS
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr. Lavrentieva 8, Novosibirsk, Russia. lavrik@niboch.nsc.ru
Khodyreva
S N
SN
Lavrik
O I
OI
D000818:Animals; D001386:Azides / Q000737:chemistry; D002457:Cell Extracts; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair; D004261:DNA Replication; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000737:chemistry; D051379:Mice; D009841:Oligonucleotides / Q000378:metabolism; D019920:Photoaffinity Labels; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D000076105:X-ray Repair Cross Complementing Protein 1
D001386:Azides; D002457:Cell Extracts; D017931:DNA Primers; D004268:DNA-Binding Proteins; D003845:Deoxycytosine Nucleotides; C529092:N-(2-N-(N-(4-azido-2,5-difluoro-3-chloropyridin-6-yl)-3-aminopropionyl)aminoethyl)-2'-deoxycytidine-5'-triphosphate; D009841:Oligonucleotides; D019920:Photoaffinity Labels; D011994:Recombinant Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; C000619352:Xrcc1 protein, mouse; D004247:DNA; D019951:DNA Polymerase beta; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase