Use of modified flap structures for study of base excision repair proteins.
To investigate interactions between proteins participating in the long-patch pathway of base excision repair (BER), DNA duplexes with flap strand containing modifications in sugar phosphate backbone within the flap-forming oligonucleotides were designed. When the flap-forming oligonucleotide consisted of two sequences bridged by a decanediol linker located in the flap strand near the branch point, the efficiency and position of cleavage by flap endonuclease 1 (FEN1) differed from those for natural flap. The cleavage rate of chimeric structure by FEN1 was lower than that of a normal substrate. When we introduced the second modification in the flap-forming oligonucleotide, the cleavage rate decreased significantly. To estimate efficiency of recognition and processing of the chimeric structures by BER proteins, we studied the rate of DNA synthesis by DNA polymerase beta (Pol beta) and the rate of nucleotide excision at the 3'-end of the initiating primer by apurinic/apyrimidinic endonuclease 1 (APE1) compared with those for the natural DNA duplexes. Efficiency of strand-displacement DNA synthesis catalyzed by Pol beta was shown to be higher for flap structures containing non-nucleotide linkers. The chimeric structures were processed by the 3'-exonuclease activity of APE1 with efficiency lower than that for a normal flap structure. Thus, DNA duplexes with modifications in sugar phosphate backbone can be used to mimic intermediates of the long-patch pathway of BER in reconstituted systems containing FEN1. Based on chimeric and natural oligonucleotides, photoreactive DNA structures were designed. The photoreactive dCMP moiety was introduced into the 3'-end of DNA primer via the activity of Pol beta. The photoreactive DNA duplexes--3'-recessed DNA, nicked DNA, and flap structures containing natural and chimeric oligonucleotides--were used for photoaffinity labeling of BER proteins.
Nazarkina
Zh K
ZhK
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia.
Pyshnyi
D V
DV
Pyshnaya
I A
IA
Lavrik
O I
OI
Khodyreva
S N
SN
D001345:Autoradiography; D001483:Base Sequence; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005092:Exonucleases / Q000378:metabolism; D045585:Flap Endonucleases / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000737:chemistry
D003854:Deoxyribonucleotides; D019920:Photoaffinity Labels; D019951:DNA Polymerase beta; D005092:Exonucleases; D045585:Flap Endonucleases; C022650:spleen exonuclease; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase