Interaction of flap endonuclease-1 and replication protein A with photoreactive intermediates of DNA repair.
A new method for enzymatic synthesis of radioactive DNA flapped structures containing a photoreactive dCMP moiety at a branch point with 4-(4-azido-2,3,5,6-tetrafluorobenzylidene-hydrazinocarbonyl)butylcarbamoyl group attached at exo-N-position of cytosine was developed. The formation of complexes of flap endonuclease-1 (FEN-1) with flapped DNA was shown by photoaffinity modification and gel retardation assays. The substrate properties of the flapped structures with different flap lengths were studied in the reaction of endonuclease cleavage catalyzed by FEN-1. It was demonstrated that inhibition of FEN-1 activity by replication protein A (RPA) depends on the length of the single-stranded part of the flapped substrate. A significant inhibition of cleavage was observed when the flap length was sufficient for effective RPA binding, while for structures with short single-stranded part the efficiency of cleavage was independent of the presence of RPA. FEN-1 and RPA were modified by photoaffinity labeling using flap structures with single-stranded parts consisting of 8 and 21 nucleotides. Products of DNA photoattachment to FEN-1 were observed in both cases, while the covalent adducts with RPA were obtained only with the 21-nucleotide-long flap. Photoaffinity modification demonstrated that FEN-1 and RPA compete for the binding of the flapped substrates with long single-stranded parts.
Nazarkina
J K
JK
Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
Petrousseva
I O
IO
Safronov
I V
IV
Lavrik
O I
OI
Khodyreva
S N
SN
D001483:Base Sequence; D004260:DNA Repair / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000201:enzymology; D045585:Flap Endonucleases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry; D019920:Photoaffinity Labels / Q000737:chemistry; D051716:Replication Protein A
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D019920:Photoaffinity Labels; D051716:Replication Protein A; D045585:Flap Endonucleases