Construction of Escherichia coli vectors containing deoxyadenosine and deoxyguanosine adducts from (+)-anti-dibenz[a,j]anthracene diol epoxide at a defined site.
Construction of Escherichia coli vectors containing deoxyadenosine and deoxyguanosine adducts from (+)-anti-dibenz[a,j]anthracene diol epoxide at a defined site.
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构建含有在指定位点的 ( )-抗二苯并[a,j]蒽二醇环氧化物的脱氧腺苷和脱氧鸟苷加合物的大肠杆菌载体。
DOI：
10.1021/tx00035a014
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发表时间：
1993
影响因子：
4.1
通讯作者：
DiGiovanni,J
中科院分区：
文献类型：
--
作者：
Gill,RD;Min,Z;Cortez,C;Harvey,RG;Loechler,EL;DiGiovanni,J
二苯并[a J]菲(Db[a J]A)是一种致癌的多环芳烃，通过海湾地区二醇环氧化物的形成而被代谢激活。构建了含有单一(+)-<；MTI-dibenz[a J]菲二醇环氧化物{(+)-<；MFI-DB[AJ]A-DE}-脱氧鸟苷(DGuo)或-脱氧腺苷(Dado)加合物的M13mpl9载体。分别对应于M13mpl9多克隆区Psf1和Spl1限制性内切酶中心4个碱基对的四碱基寡核苷酸S‘-hoTGCA-S’和5‘-hoCATG-3’分别与(ą)-ANFI-DB[AJ]A-DE反应。用反相高效液相色谱分离纯化得到的加合寡核苷酸。分离到了几种不同的单加成寡核苷酸，包括与dGuo或dado结合的二醇环氧化物的(+)和(-)对映体的不同顺式和反式加成产物。选择含有(+)-抗DB[AJ]A-Trans-N2*-dGuo加合物[T(DB[AJ]A-N2)GCA]的5‘-hoTGCA-3’和含有(+)-抗DB[AJ]A-tran-N6-Dado加合物[C(DB[AJ]A-N6)ATG)的5‘-hoCATG-3’连接到已构建的M13mpl9载体中，该载体在负链上具有对应的四个碱基间隙。未修饰和加合的寡核苷酸均成功地连接到M13mpl9载体中，[产率：未修饰的TGCA-M13mpl9(~32%)和-CATG-M13mpl9(~42%)；加合物T(DB[AJ]A-N2)GCA-M13mpl9(~13%)和C(DB[AJ]A-N6)ATG-M13mpl9(~12%)]。对含有DAO加合物的载体进行了表征。在SPHL的识别位点上存在DADO-DNA加合物可抑制SPHL的限制。含有DADO加合物的载体被Hindlll/Xbal切割以释放24聚体中的加合物，其迁移速度慢于从未经修饰的载体释放的相应的24聚体。含有dGuo加合物的载体也有类似的特征。虽然结果比较复杂，但大约25%的载体含有dGuo加合物。这些定点修饰的载体将用于未来的体外和体内研究，以比较dado和dguo加合物对复制、修复和突变的影响。
Dibenz [a J] anthracene (DB [a J] A) is a carcinogenic polycyclic aromatic hydrocarbon, which is metabolically activated through the formation of bay region diol epoxides. Site-specifically modified M13mpl9-based vectors containing a single (+)-< mti-dibenz [a J] anthracene diol epoxide {(+)-< mfi-DB [aJ] A-DE}-deoxyguanosine (dGuo) or-deoxyadenosine (dAdo) adduct were constructed. Four-base oligonucleotides, S'-hoTGCA-S'and 5'-hoCATG-3', corresponding to the central four base pairs in the Psfl and Sphl restriction endonuclease sites, respectively, in the multiple cloning region of M13mpl9, were reacted in solution with (ą)-anfi-DB [aJ] A-DE. The resulting adducted oligonucleotides were separated and purified using reverse-phase HPLC. Several different singly adducted oligonucleotides were isolated, consisting of the various cis and trans addition products of the (+) and (-) enantiomers of the diol epoxide bound to dGuo or dAdo in the oligonucleotides. 5'-hoTGCA-3'containing the (+)-anti-DB [aJ] A-trans-N2*-dGuo adduct [T (DB [aj] A-N2) GCA] and 5'-hoCATG-3'containing the (+)-anti-DB [aj] A-trans-N6-dAdo adduct [C (DB [aj] A-N6) ATG) were selected for subsequent ligation into M13mpl9 vectors that had been constructed with a corresponding four base gap in the minus strand. Both unmodified and adducted oligonucleotides were successfully ligated into the M13mpl9 vectors,[yields: unmodified-TGCA-M13mpl9 (~ 32%) and-CATG-M13mpl9 (~ 42%); adducted T (DB [aj] A-N2) GCA-M13mpl9 (~ 13%) and C (DB [aj] A-N6) ATG-M13mpl9 (~ 12%)]. The dAdo adduct-containing vector was characterized. The presence of a dAdo-DNA adduct at the recognition site of Sphl inhibited restriction by Sphl. The dAdo adduct-containing vector was cleaved with Hindlll/Xbal to release the adduct in a 24-mer, which migrated slower than the corresponding 24-merreleased from the unmodified vector. The dGuo adduct-containing vector was characterized similarly. While the results were more complicated, approximately 25% of the vectors contained the dGuo adduct. These site-specifically modified vectors will be used in future studies in vitro and in vivo to compare the effects of dAdo vs dGuo adducts on replication, repair, andmutagenesis.