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| - Preferential Repair Incision of Cross-Links versus Monoadducts inPsoralen-Damaged Plasmid DNA by Human Cell-Free Extracts
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| - Upon UVA irradiation psoralens covalently bind to DNA asmonoadduct and interstrand cross-link. Psoralen photoadducts are processed via anexcision repair reaction that has been reproduced invitro with transcriptionnally active cell-free extracts. Aderived in vitro assay that allows directquantification of the incised sites has been set up and used to comparethe efficiency of the incisionreaction on monoadducts and interstrand cross-links. The incisionreaction was performed with HeLacell-free extracts on angelicin or 8-methoxypsoralen (8-MOP)-modifiedplasmid DNA substrates carryingknown amounts of mono- and biadducts, within various relative ratios.In the case of 8-MOP modifiedplasmids consisting in a mixture of mono- and biadducts on the same DNAmolecule, the incision signalwas mainly due to the presence of interstrand cross-links. Theextent of incision was linear with thenumber of cross-links up to about 4 cross-links per plasmid and thenreached a plateau. The sensitivityof incision defined as the increase of incision by 2-fold over thebackground level corresponded to about1 cross-link per plasmid molecule, and about 7% of the totalcross-links were repaired under our assayconditions. The incision activity on angelicin monoadducts yieldedonly 27% when compared to that on8-MOP cross-links. Furthermore, 8-MOP cross-links lowered theincision extent of angelicin monoadductswhen the two photoadducts were present on distinct plasmid DNAmolecules. These data are in line withthe more rapid excision of psoralen interstrand cross-links vsmonoadducts observed in vivo.
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