Ide H, Wallace S S
Department of Microbiology and Immunology, New York Medical College, Valhalla 10595.
Nucleic Acids Res. 1988 Dec 9;16(23):11339-54. doi: 10.1093/nar/16.23.11339.
The ability of dihydrothymidine (DHdTTP) and thymidine glycol (dTTP-GLY) 5'-triphosphates to serve as substrates for different DNA polymerases was investigated. DHdTTP but not dTTP-GLY was used as a substrate by E. coli DNA polymerase I (Pol I). Within the detection limit of the assay used, neither T4 DNA polymerase nor avian myeloblastosis virus (AMV) reverse transcriptase used DHdTTP or dTTP-GLY as substrates. The ability of DHdTTP and dTTP-GLY to undergo enzyme-catalyzed turnover to the monophosphate paralleled their ability to serve as substrates for polymerization. These results, along with kinetic parameters for the incorporation of DHdTTP with Pol I, strongly suggest that the saturation of thymine C5-C6 bond and the substituent groups at C5 and C6 differentially exert effects on binding to DNA polymerases. DNA sequencing gel analysis of the polymerization products revealed that most single adenine sites were capable of templating DHdTTP, however, DNA synthesis was partially arrested at multiple adenine sites, suggesting that sequential incorporation of DHdTTP produced significant disorder in the primer terminus.
研究了二氢胸苷三磷酸（DHdTTP）和胸苷二醇三磷酸（dTTP - GLY）作为不同DNA聚合酶底物的能力。大肠杆菌DNA聚合酶I（Pol I）使用DHdTTP而非dTTP - GLY作为底物。在所使用检测方法的检测限内，T4 DNA聚合酶和禽成髓细胞瘤病毒（AMV）逆转录酶均未将DHdTTP或dTTP - GLY用作底物。DHdTTP和dTTP - GLY经酶催化转化为单磷酸的能力与其作为聚合反应底物的能力平行。这些结果，连同DHdTTP与Pol I掺入的动力学参数，强烈表明胸腺嘧啶C5 - C6键的饱和以及C5和C6处的取代基对与DNA聚合酶的结合有不同影响。对聚合产物的DNA测序凝胶分析表明，大多数单个腺嘌呤位点能够作为DHdTTP的模板，然而，DNA合成在多个腺嘌呤位点部分停滞，这表明DHdTTP的连续掺入在引物末端产生了明显的紊乱。
