Highly selective affinity labeling of DNA-polymerase from Thermus thermophilus B35 by a binary system of photoreactive agents.
The thermostable DNA-polymerase from Thermus thermophilus B35 (Tte-polymerase) was affinity labeled by a binary system of photoreagents comprising base-substituted TTP analogs. The 5;-[32P]-labeled primer was elongated by Tte-polymerase in the presence of a TTP analog containing the photoreactive 2,3,5, 6-tetrafluoro-4-azidobenzoyl group (FAB-4-dUTP). Then the reaction mixture was UV-irradiated (365-450 nm) in the presence or the absence of a photosensitizer (TTP analog containing a pyrene moiety, Pyr-dUTP). The initial rate of the Pyr-dUTP-sensitized photomodification was almost 10-fold higher than the rate of direct photomodification (in the absence of Pyr-dUTP); in the case of the sensitized modification, the product of covalent cross-linking of the photoreactive primer with Tte-polymerase was apparently homogenous according to the data of electrophoresis. The enzyme was protected from the photosensitized modification by dNTP. To confirm the selectivity of the photosensitized modification of Tte-polymerase, another DNA-binding protein (human replication factor A, RPA) was added to the reaction mixture. In the presence of the photosensitizer (Pyr-dUTP), RPA was not labeled and only Tte-polymerase was modified, whereas in the case of direct modification, Tte-polymerase and the p32 and p70 subunits of RPA were labeled. The suggested method enables highly selective affinity modification of DNA-polymerases.
Rechkunova
N I
NI
Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia. nadyarec@niboch.nsc.ru.
Kolpashchikov
D M
DM
Lebedeva
N A
NA
Petruseva
I O
IO
Dobrikov
M I
MI
Degtyarev
S K
SK
Lavrik
O I
OI
D000345:Affinity Labels; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D017319:Photosensitizing Agents / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A; D016963:Thermus thermophilus / Q000201:enzymology*; D013942:Thymine Nucleotides / Q000494:pharmacology
C119021:5-(N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)aminopropenyl-1)-2'-deoxyuridine-5'-triphosphate; C119022:5-(N-(4-(1-pyrenyl)butylcarbonyl)aminopropenyl-1)-2'-deoxyuridine-5'-triphosphate; D000345:Affinity Labels; D017931:DNA Primers; D004268:DNA-Binding Proteins; D003856:Deoxyuracil Nucleotides; D017319:Photosensitizing Agents; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A; D013942:Thymine Nucleotides; D004259:DNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate