Mechanism of action of deoxyribonuclease II from human lymphoblasts.
Mechanism of action of deoxyribonuclease II from human lymphoblasts.
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人淋巴母细胞脱氧核糖核酸酶 II 的作用机制。
DOI：
10.1111/j.1432-1033.1991.tb16397.x
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Boyd,JB
中科院分区：
文献类型：
--
作者：
Harosh,I;Binninger,DM;Harris,PV;Mezzina,M;Boyd,JB
从人淋巴母细胞K562中分离纯化了脱氧核糖核酸酶II。分离包括DEAE -纤维素和肝素-琼脂糖层析，然后在Mono - S， Mono - Q和Superose - 12 FPLC柱上进行分离。在先前研究的延伸中，研究人员发现，脱氧核糖核酸酶II在超螺旋DNA中引入的单链缺口比双链断裂的比例要高得多。DNA测序技术的应用进一步揭示了3 '末端对该酶水解的独特抗性。脱氧核糖核酸酶II沿着一对寡核苷酸底物的双工部分在每个可用的位点上切割，最后四个核苷酸除外。与之前的结果一致，该脱氧核糖核酸酶II在低pH下没有Mg2+时具有活性，并且不受EDTA的抑制，但在100 μM Fe3+中观察到完全抑制。同样，我们证实了DNA切割产物上存在3′‐磷酸化末端，因为它们不能作为引物用于大肠杆菌DNA聚合酶I催化的DNA合成。
Deoxyribonuclease II has been purified through five fractionation steps from the human lymphoblast cell line K562. Isolation included DEAE‐cellulose and heparin – agarose chromatography followed by fractionation on Mono‐S, Mono‐Q and Superose‐12 FPLC columns. In an extension of previous studies, deoxyribonuclease II was found to introduce a much higher proportion of single‐strand nicks relative to double‐strand breaks into supercoiled DNA than has been reported for linear DNA. Application of DNA sequencing techniques has further revealed a unique resistance of 3′ termini to hydrolysis by this enzyme.Deoxyribonuclease II cleaves at every available site along the duplexed portion of a paired oligonucleotide substrate with the exception of the last four nucleotides. Consistent with previous results, this deoxyribonuclease II is active at low pH in the absence of Mg2+and is not inhibited by EDTA, but complete inhibition is observed with 100 μM Fe3+. Likewise we confirmed the presence of 3′‐phosphoryl termini on the DNA cleavage products since they failed to function as primers for DNA synthesis catalyzid byEscherichia coliDNA polymerase I.