Shibutani S, Bodepudi V, Johnson F, Grollman A P
Department of Pharmacological Sciences, State University of New York, Stony Brook 11794-8651.
Biochemistry. 1993 May 4;32(17):4615-21. doi: 10.1021/bi00068a019.
This study was designed to establish the miscoding potential of 8-oxo-7,8-dihydrodeoxyadenosine (8-oxo-dA). Oligodeoxynucleotides modified site-specifically with 8-oxo-dA were used as templates in primer extension reactions catalyzed by DNA polymerase I (Klenow fragment), DNA polymerase alpha (pol alpha), or DNA polymerase beta (pol beta). dTMP or dGMP is incorporated opposite 8-oxo-dA when either of these dNTPs is provided as substrate for DNA polymerase. dTMP is incorporated exclusively opposite 8-oxo-dA when all four dNTPs are present in the reaction mixture at equimolar concentrations. Chain extension is catalyzed efficiently by Klenow fragment and pol beta under conditions where 8-oxo-dA is paired with dT at the 3' terminus of the primed DNA template. Chain extension catalyzed by pol alpha proceeds more slowly. As shown by steady-state kinetic experiments, incorporation of dGMP is higher in reactions catalyzed by pol beta than by Klenow fragment or pol alpha. The dG-8-oxo-dA pair is extended efficiently from the 3' terminus in the absence of dTTP. We conclude that DNA containing 8-oxo-dA is capable of miscoding; however, unlike 8-oxo-dG, the mutagenic potential of this lesion is limited.
本研究旨在确定8-氧代-7,8-二氢脱氧腺苷（8-氧代-dA）的错编码潜力。用8-氧代-dA进行位点特异性修饰的寡脱氧核苷酸在DNA聚合酶I（克列诺片段）、DNA聚合酶α（polα）或DNA聚合酶β（polβ）催化的引物延伸反应中用作模板。当这些dNTP中的任何一种作为DNA聚合酶的底物时，dTMP或dGMP会掺入到与8-氧代-dA相对的位置。当反应混合物中所有四种dNTP以等摩尔浓度存在时，dTMP仅掺入到与8-氧代-dA相对的位置。在8-氧代-dA与引发的DNA模板3'末端的dT配对的条件下，克列诺片段和polβ能有效地催化链延伸。polα催化的链延伸进行得较慢。稳态动力学实验表明，polβ催化的反应中dGMP的掺入量高于克列诺片段或polα催化的反应。在没有dTTP的情况下，dG-8-氧代-dA对能从3'末端有效地延伸。我们得出结论，含有8-氧代-dA的DNA能够错编码；然而，与8-氧代-dG不同，这种损伤的诱变潜力是有限的。
