Calsou P, Sage E, Moustacchi E, Salles B
Institut de Pharmacologie et Biologie Structurale, UPR 9062 CNRS, Toulouse, France.
Biochemistry. 1996 Nov 26;35(47):14963-9. doi: 10.1021/bi9607261.
Upon UVA irradiation psoralens covalently bind to DNA as monoadduct and interstrand crosslink. Psoralen photoadducts are processed via an excision repair reaction that has been reproduced in vitro with transcriptionnally active cell-free extracts. A derived in vitro assay that allows direct quantification of the incised sites has been set up and used to compare the efficiency of the incision reaction on monoadducts and interstrand cross-links. The incision reaction was performed with HeLa cell-free extracts on angelicin or 8-methoxypsoralen (8-MOP)-modified plasmid DNA substrates carrying known amounts of mono- and biadducts, within various relative ratios. In the case of 8-MOP modified plasmids consisting in a mixture of mono- and biadducts on the same DNA molecule, the incision signal was mainly due to the presence of interstrand cross-links. The extent of incision was linear with the number of cross-links up to about 4 cross-links per plasmid and then reached a plateau. The sensitivity of incision defined as the increase of incision by 2-fold over the background level corresponded to about 1 cross-link per plasmid molecule, and about 7% of the total cross-links were repaired under our assay conditions. The incision activity on angelicin monoadducts yielded only 27% when compared to that on 8-MOP cross-links. Furthermore, 8-MOP cross-links lowered the incision extent of angelicin monoadducts when the two photoadducts were present on distinct plasmid DNA molecules. These data are in line with the more rapid excision of psoralen interstrand cross-links vs monoadducts observed in vivo.
在紫外线A照射下，补骨脂素以单加合物和链间交联的形式与DNA共价结合。补骨脂素光加合物通过一种切除修复反应进行处理，这种反应已在具有转录活性的无细胞提取物中体外重现。已经建立了一种衍生的体外测定方法，可直接定量切割位点，并用于比较单加合物和链间交联上切割反应的效率。切割反应是用HeLa细胞无细胞提取物对携带已知量单加合物和双加合物的当归素或8-甲氧基补骨脂素（8-MOP）修饰的质粒DNA底物进行的，比例各不相同。在同一DNA分子上由单加合物和双加合物混合物组成的8-MOP修饰质粒的情况下，切割信号主要归因于链间交联的存在。切割程度与交联数量呈线性关系，直至每个质粒约4个交联，然后达到平稳期。定义为切割量比背景水平增加2倍的切割敏感性相当于每个质粒分子约1个交联，在我们的测定条件下，约7%的总交联被修复。与8-MOP交联相比，当归素单加合物的切割活性仅为27%。此外，当两种光加合物存在于不同的质粒DNA分子上时，8-MOP交联降低了当归素单加合物的切割程度。这些数据与体内观察到的补骨脂素链间交联比单加合物切除更快一致。
