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PHOTOADDITION OF 8‐METHOXYPSORALEN TO E. coli DNA POLYMERASE I. ROLE OF PSORALEN PHOTO‐ADDUCTS IN THE PHOTOSENSITIZED ALTERATIONS OF POL I ENZYMATIC ACTIVITIES
Author(s) -
Granger Michele,
Héène Claude
Publication year - 1983
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1983.tb03384.x
Subject(s) - psoralen , chemistry , singlet oxygen , photochemistry , oxygen , reactive oxygen species , covalent bond , enzyme , adduct , polymerase , dna , biochemistry , organic chemistry
— We have previously demonstrated that 8‐methoxypsoralen (8‐MOP)‡ plus UVA is able to inactivate the three enzymatic activities of E. coli DNA polymerase I and that oxygen is required for these reactions (M. Granger et al. , (1982) Photochem. Photobiol. , 36 , 175–180). We now show that UV‐A irradiation produces a covalent incorporation of the psoralen derivative into the enzyme either in the presence or in the absence of oxygen. The excited psoralen binds directly to the protein in an oxygen‐independent reaction; no complex was detected in the absence of irradiation. Fluorescence measurements reveal that at least two photoadducts are formed. The 8‐MOP‐photomodified enzyme is still fully active but further irradiation leads to an inhibition of the 5′→ 3′ polymerase activity whereas the 5′→ 3′ exonuclease activity is not affected. A major part of the inhibition reaction is shown to be oxygen‐dependent but singlet oxygen quenchers have no effect on the kinetics. This oxygen‐dependent reaction is attributed to a photosensitization, due to covalently bound 8‐MOP, of neighbouring amino acids through an intermediate reactive oxygen species which is not singlet oxygen. The oxygen‐independent reaction is attributed to a direct photosensitization through, for example, a radical mechanism.