Sensitized photomodification of mammalian DNA polymerase beta. A new approach for highly selective affinity labeling of polymerases.
To enhance the specificity of polymerase photoaffinity labeling, a novel approach based on sensitized photomodification has been developed. A base-substituted analog of TTP containing a pyrene group (PyrdUTP) was synthesized and used as an active site-bound photosensitizer for photoaffinity modification of DNA polymerase beta (pol beta). 5'-[32P]-labeled primer was elongated in situ by pol beta with a photoreactive analog of TTP (FAB-4-dUTP). The pyrene sensitizer (PyrdUTP), excited by light (365-450 nm), can activate the photoreagent, cross-linking it to pol beta as a result of fluorescence resonance energy transfer. The initial rate of pol beta photomodification was shown to increase by a factor of ten. The selectivity of pol beta photosensitized modification was proved by adding human replication protein A.
Kolpashchikov
D M
DM
Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences.
Rechkunova
N I
NI
Dobrikov
M I
MI
Khodyreva
S N
SN
Lebedeva
N A
NA
Lavrik
O I
OI
D000818:Animals; D019951:DNA Polymerase beta / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D006801:Humans; D008322:Mammals; D015394:Molecular Structure; D010441:Peptide Chain Elongation, Translational; D019920:Photoaffinity Labels / Q000378:metabolism*; D017319:Photosensitizing Agents / Q000378:metabolism*; D051716:Replication Protein A; D013379:Substrate Specificity
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; D004268:DNA-Binding Proteins; D003856:Deoxyuracil Nucleotides; D019920:Photoaffinity Labels; D017319:Photosensitizing Agents; C497167:RPA1 protein, human; D051716:Replication Protein A; C027078:deoxyuridine triphosphate; D019951:DNA Polymerase beta