含有2',3'-稠合三元环的修饰核苷5'-三磷酸作为不同DNA聚合酶的底物。

Modified nucleoside 5'-triphosphates containing 2',3'-fused three-membered rings as substrates for different DNA polymerases.

作者信息

Semizarov D G, Victorova L S, Krayevsky A A, Kukhanova M K

机构信息

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.

出版信息

FEBS Lett. 1993 Jul 19;327(1):45-8. doi: 10.1016/0014-5793(93)81036-y.

DOI:10.1016/0014-5793(93)81036-y

PMID:7687565

Abstract

5'-Triphosphates of 1-(2',3'-epithio-2',3'-dideoxy-beta-D- lyxofuranosyl)thymine, 1-(2',3'-epithio-2',3'-dideoxy-beta-D-ribofuranosyl)thymine and 2',3'-lyxoanhydrothymidine have been shown to be termination substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) reverse transcriptases as well as DNA polymerase I from E. coli and DNA polymerase beta from rat liver. At the same time they do not terminate DNA synthesis catalysed by DNA polymerase epsilon from human placenta. Km values of ltTTP, rtTTP and laTTP incorporation into the DNA chain during catalysis by AMV reverse transcriptase agree closely with each other being 1.5-2.5 times higher than Km value for dTTP. Furthermore, Vmax values for modified substrates are only 2-3 times lower than Vmax for dTTP. The evidence favours the hypothesis of high affinity of modified nucleotides with a flattened furanosyl ring for DNA polymerase active sites.

摘要

1-(2',3'-环氧-2',3'-二脱氧-β-D-来苏呋喃糖基)胸腺嘧啶、1-(2',3'-环氧-2',3'-二脱氧-β-D-核糖呋喃糖基)胸腺嘧啶和2',3'-来苏缩醛胸腺嘧啶的5'-三磷酸已被证明是人类免疫缺陷病毒(HIV)、禽成髓细胞瘤病毒(AMV)逆转录酶以及大肠杆菌DNA聚合酶I和大鼠肝脏DNA聚合酶β的终止底物。同时，它们不会终止人胎盘DNA聚合酶ε催化的DNA合成。在AMV逆转录酶催化过程中，ltTTP、rtTTP和laTTP掺入DNA链的Km值彼此非常接近，比dTTP的Km值高1.5-2.5倍。此外，修饰底物的Vmax值仅比dTTP的Vmax值低2-3倍。证据支持具有扁平呋喃糖环的修饰核苷酸对DNA聚合酶活性位点具有高亲和力的假说。