Jo K, Topal M D
Lineberger Comprehensive Cancer Center and Department of Pathology, University of North Carolina Medical School, Chapel Hill 27599-7295, USA.
Nucleic Acids Res. 1996 Nov 1;24(21):4171-5. doi: 10.1093/nar/24.21.4171.
Substituting lysine for leucine at position 43 (L43K) transforms NaeI from restriction endonuclease to topoisomerase and makes NaeI hypersensitive to intercalative anticancer drugs. Here we investigated DNA recognition by Nael-L43K. Using DNA competition and gel retardation assays, NaeI-L43K showed reduced affinity for DNA substrate and the ability to bind both single- and double-stranded DNA with a definite preference for the former. Sedimentation studies showed that under native conditions NaeI-L43K, like NaeI, is a dimer. Introduction of mismatched bases into double-stranded DNA significantly increased that DNA's ability to inhibit NaeI-L43K. Wild-type NaeI showed no detectable binding of either single-stranded DNA or mismatched DNA over the concentration range studied. These results demonstrate that the L43K substitution caused a significant change in recognition specificity by NaeI and imply that NaeI-L43K's topoisomerase activity is related to its ability to bind single-stranded and distorted regions in DNA. A mechanism is proposed for the evolution of the NaeI restriction-modification system from a topoisomerase/ligase by a mutation that abolished religation activity and provided a needed change in DNA recognition.
将第43位的亮氨酸替换为赖氨酸（L43K）可使NaeI从限制性内切核酸酶转变为拓扑异构酶，并使NaeI对嵌入性抗癌药物高度敏感。在此，我们研究了Nael-L43K对DNA的识别。通过DNA竞争和凝胶阻滞试验，NaeI-L43K对DNA底物的亲和力降低，并且能够结合单链和双链DNA，对前者有明确的偏好。沉降研究表明，在天然条件下，NaeI-L43K与NaeI一样，是一种二聚体。将错配碱基引入双链DNA显著增强了该DNA抑制NaeI-L43K的能力。在研究的浓度范围内，野生型NaeI对单链DNA或错配DNA均未表现出可检测到的结合。这些结果表明，L43K替换导致NaeI的识别特异性发生了显著变化，并暗示NaeI-L43K的拓扑异构酶活性与其结合DNA中单链和扭曲区域的能力有关。我们提出了一种机制，说明NaeI限制修饰系统是如何从一种拓扑异构酶/连接酶通过一个消除重新连接活性并在DNA识别方面产生所需变化的突变进化而来的。
