Bedows E, Wachsman J T, Gumport R I
Biochemistry. 1977 May 17;16(10):2231-5. doi: 10.1021/bi00629a029.
L cell DNA ligase catalyzes a covalent linkage between 5'-hosphoryl oligodeoxyribonucleotides and 3'-hydroxyl oligoribonucleotides on a complementary polydeoxyribonucleotide template. This reaction occurs to a substantially lesser extent than does the sealing of DNA to DNA. The joining of [5'32P]d(pA)12-18 to (Ap)11A on poly[d(T)] or of [5'-32P]d(pG)12-18 to 5'-hydroxyl, 3'-hydroxyl oligo(I) ON POLY[D(C)] was demonstrated by the formation of alkaline phosphatase resistant radioactivity. The 32P of the hybrid reaction products became sensitive to the action of alkaline phosphatase after treatment with alkali. Furthermore, hydrolysis of the products of the linkage of [5'-32P]d(pG)12-18 to 5'-hydroxyl, 3'-hydroxyl oligo(I) on poly[d(C)] with micrococcal nuclease and spleen phosphodiesterase resulted in the formation of [3'-32P]IMP. Attempts to seal [5'-32p[-(pA)12 to d(Ap)11-17A on poly[d(T) or [5'-32P]oligo(pI) to d(Gp)11-17G on poly[d(C)] were unsuccessful.
L细胞DNA连接酶催化在互补的聚脱氧核糖核苷酸模板上，5'-磷酸化寡聚脱氧核糖核苷酸与3'-羟基寡聚核糖核苷酸之间形成共价连接。与DNA与DNA的封闭反应相比，该反应发生的程度要小得多。通过形成对碱性磷酸酶有抗性的放射性，证明了[5'32P]d(pA)12-18与聚[d(T)]上的(Ap)11A或[5'-32P]d(pG)12-18与聚[d(C)]上的5'-羟基、3'-羟基寡聚(I)的连接。用碱处理后，杂交反应产物的32P对碱性磷酸酶的作用变得敏感。此外，用微球菌核酸酶和脾磷酸二酯酶水解[5'-32P]d(pG)12-18与聚[d(C)]上的5'-羟基、3'-羟基寡聚(I)的连接产物，导致形成[3'-32P]IMP。试图封闭聚[d(T)]上的[5'-32p[-(pA)12与d(Ap)11-17A或聚[d(C)]上的[5'-32P]寡聚(pI)与d(Gp)11-17G均未成功。
