Hentosh P, McCastlain J C, Blakley R L
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Biochemistry. 1991 Jan 15;30(2):547-54. doi: 10.1021/bi00216a032.
2-Chloro-2'-deoxyadenosine 5'-triphosphate (CldATP) was compared with dATP as a substrate for DNA synthesis by bacterial and viral DNA polymerases in vitro. Lengths of chain extension and DNA synthesis pause sites were determined by comparison with products generated by dideoxynucleotide sequencing methods on the same end-labeled primer/template duplex after high-resolution polyacrylamide gel electrophoresis. Reverse transcriptase (RT) from human immunodeficiency virus (HIV-1) and avian myeloblastosis virus (AMV) incorporated CldATP efficiently. DNA strand elongation continued past most chloroadenine (ClA) insertion sites but resulted in shorter chains than when dATP was inserted. Phage T4 DNA polymerase incorporated CldATP least efficiently; Klenow fragment of Escherichia coli DNA polymerase I and modified T7 DNA polymerase (Sequenase) showed intermediate ability to utilize the analogue. Incorporation of several consecutive ClA residues into the replicating strand dramatically reduced the ability of Sequenase, Klenow fragment, and T4 DNA polymerases to continue strand elongation. In the absence of the corresponding normal deoxyribonucleoside triphosphate during DNA synthesis, ClA was frequently misincorporated as thymine, cytosine, or guanine by both AMV RT and HIV-1 RT but rarely, if at all, by Klenow fragment, Sequenase, and T4 DNA polymerase. Except T4, for most DNA polymerases, CldATP at 10-20-fold molar excess over dATP was not a strong competitive inhibitor of dATP, as judged by the amount of strand extension and polymerase pause sites during DNA synthetic reactions. Our results indicate that the degree of strand extension in the presence of CldATP, the number and location of polymerase pause sites, and the amount of misincorporation of the analogue are both polymerase- and sequence-dependent.
在体外，将2-氯-2'-脱氧腺苷5'-三磷酸（CldATP）与脱氧腺苷三磷酸（dATP）作为细菌和病毒DNA聚合酶进行DNA合成的底物进行比较。通过在高分辨率聚丙烯酰胺凝胶电泳后，与在相同末端标记的引物/模板双链体上通过双脱氧核苷酸测序方法产生的产物进行比较，确定链延伸长度和DNA合成暂停位点。来自人类免疫缺陷病毒（HIV-1）和禽成髓细胞瘤病毒（AMV）的逆转录酶（RT）能有效地掺入CldATP。DNA链延伸在大多数氯腺嘌呤（ClA）插入位点之后继续，但产生的链比插入dATP时短。噬菌体T4 DNA聚合酶掺入CldATP的效率最低；大肠杆菌DNA聚合酶I的Klenow片段和修饰的T7 DNA聚合酶（测序酶）显示出利用该类似物的中等能力。在复制链中掺入几个连续的ClA残基会显著降低测序酶、Klenow片段和T4 DNA聚合酶继续链延伸的能力。在DNA合成过程中，若不存在相应的正常脱氧核糖核苷三磷酸，AMV RT和HIV-1 RT都会频繁地将ClA错掺入胸腺嘧啶、胞嘧啶或鸟嘌呤中，但Klenow片段、测序酶和T4 DNA聚合酶极少（如果有的话）出现这种情况。除了T4，对于大多数DNA聚合酶而言，根据DNA合成反应期间的链延伸量和聚合酶暂停位点判断，摩尔过量10 - 20倍的CldATP并非dATP的强竞争性抑制剂。我们的结果表明，在CldATP存在下的链延伸程度、聚合酶暂停位点的数量和位置以及该类似物的错掺入量均取决于聚合酶和序列。
