Repair of DNA strand gaps and nicks containing 3′-phosphate and 5′-hydroxyl termini by purified mammalian enzymes
Repair of DNA strand gaps and nicks containing 3′-phosphate and 5′-hydroxyl termini by purified mammalian enzymes
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DOI：
10.1093/nar/26.19.4395
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发表时间：
1998-10-01
影响因子：
14.9
通讯作者：
Weinfeld, M
中科院分区：
文献类型：
--
作者：
Karimi-Busheri, F;Lee, J;Weinfeld, M
具有5 ′-磷酸转移酶和3 ′-磷酸酶活性的哺乳动物多核苷酸激酶的推定作用是将具有5 ′-羟基末端或3 ′-磷酸末端或两者的DNA链断裂恢复为支持DNA修复聚合酶和DNA连接酶的后续作用的形式，即5 ′-磷酸和3 ′-羟基末端。为了进一步评估这种可能性，我们比较了纯化的小牛胸腺多核苷酸激酶的3 ′-磷酸酶对各种底物的活性。观察到从双链DNA中的切口或短（1 nt）缺口位点去除3 ′-磷酸基团的速率与从单链底物去除3 ′-磷酸基团的速率相似，因此，多核苷酸激酶的这种活性似乎不受磷酸基团的空间可及性的影响。我们随后证明，多核苷酸激酶和纯化的人DNA连接酶I的协同反应可以有效地修复具有3 ′-磷酸和5 ′-羟基末端的DNA切口，并且类似地，这两种酶与纯化的大鼠DNA聚合酶β一起的组合可以密封具有1 nt缺口的链断裂。在多核苷酸激酶和ATP存在下，聚合酶β的缺口填充速率显著增强，表明5 ′-磷酸化的积极影响。通过向反应混合物中加入DNA连接酶I进一步增强反应。这至少部分是由于DNA连接酶I增强了多核苷酸激酶催化的5 '-磷酸化速率。
A putative role for mammalian polynucleotide kinases that possess both 5'-phosphotransferase and 3'-phosphatase activity is the restoration of DNA strand breaks with 5'-hydroxyl termini or 3'-phosphate termini, or both, to a form that supports the subsequent action of DNA repair polymerases and DNA ligases, i.e. 5'-phosphate and 3'-hydroxyl termini, To further assess this possibility, we compared the activity of the 3'-phosphatase of purified calf thymus polynucleotide kinase towards a variety of substrates, The rate of removal of 3'-phosphate groups from nicked or short (1 nt) gapped sites in double-stranded DNA was observed to be similar to that of 3'-phosphate groups from single-stranded substrates, Thus this activity of polynucleotide kinase does not appear to be influenced by steric accessibility of the phosphate group. We subsequently demonstrated that the concerted reactions of polynucleotide kinase and purified human DNA ligase I could efficiently repair DNA nicks possessing 3'-phosphate and 5'-hydroxyl termini, and similarly the combination of these two enzymes together with purified rat DNA polymerase beta could seal a strand break with a 1 nt gap, With a substrate containing a nick bounded by 3'- and 5'-OH termini, the rate of gap filling by polymerase beta was significantly enhanced in the presence of polynucleotide kinase and ATP, indicating the positive influence of 5'-phosphorylation. The reaction was further enhanced by addition of DNA ligase I to the reaction mixture. This is due, at least in part, to an enhancement by DNA ligase I of the rate of 5'-phosphorylation catalyzed by polynucleotide kinase.