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Abstract Irradiation by UV-A of E. coli DNA polymerase I in the presence of 8-methoxypsoralen (8-MOP) leads to a similar inactivation of the 5′[RIGHTWARDS ARROW] 3’polymerase and 3’[RIGHTWARDS ARROW] 5’exonuclease activities of the enzyme. The kinetics of inactivation depend on the psoralen concentration and on the duration of irradiation. The 5′[RIGHTWARDS ARROW]3’exonuclease activity is first slightly stimulated at short irradiation time in the presence of 8-MOP; further irradiation leads to inhibition of this activity. The mechanism of the reactions involves oxygen as shown by the absence of any effect of UV irradiation when oxygen is removed. The results obtained in D2O, in oxygen-saturated buffer or in the presence of singlet oxygen (1O2) quenchers show that 1O2 is one of the reactive intermediate species. Irradiation of homopolymers primed with oligomers in the presence of 8-MOP inhibits their template and initiating capacity in the same dose range as that required to inactivate DNA polymerase I. Mono-adducts of 8-MOP with nucleic acid bases can act as inhibitors of both replication and initiation.