Efficiency of T4 DNA ligase-catalyzed end joining after S1 endonuclease treatment on duplex DNA containing single-stranded portions

Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis Pub Date : 1981-11-27 DOI:10.1016/0005-2787(81)90035-6

Kazuo Shishido, Tadahiko Ando

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引用次数: 10

Abstract

Covalently closed-circular, superhelical SV40 DNA was used in all experiments. EcoRI endonuclease- and HpaII enonuclease-generated unit-length linear duplex DNAs were digested with S₁ endonuclease under the conditions where single-stranded DNA was completely converted into the acid-soluble form. These were subjected to an end-to-end joining test with T4 DNA ligase. The ligation efficiency was significantly lower than that of the flush-ended linear duplex DNAs which were generated by both HpaI endonuclease digestion and the matching up of EcoRI-generated sticky end with Escherichia coli DNA polymerase I (Klenow fraction). However, the ligation efficiency of the S₁-treated DNAs increased up to same level as the flush-ended DNA upon treatment with E. coli DNA polymerase I. Similar results were obtained in the case of S₁-generated unit-length linear duplex DNA. S₁ does cleave both strands of superhelical DNA at unbasepaired sites.

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S1内切酶处理含单链部分的双链DNA后，T4 DNA连接酶催化末端连接的效率

所有实验均采用共价封闭的超螺旋SV40 DNA。在单链DNA完全转化为酸溶形式的条件下，用S1内切酶酶切EcoRI内切酶和HpaII内切酶生成的单位长度线性双链DNA。这些都进行了T4 DNA连接酶的端到端连接试验。与HpaI酶切和ecori粘端与大肠杆菌DNA聚合酶I (Klenow分数)匹配制备的同源双链DNA相比，其连接效率显著降低。然而，用大肠杆菌DNA聚合酶i处理后，经s1处理的DNA的连接效率提高到与冲洗端DNA相同的水平，在s1产生的单位长度线性双工DNA的情况下获得了类似的结果。S1确实会在未碱基修复的位置切割超螺旋DNA的两条链。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis

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