盐对真核生物DNA缺口闭合酶与DNA和染色质结合的影响

Betty L. McConaughy, Lisa S. Young , James J. Champoux
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引用次数: 56

摘要

在DNA过量的条件下,大鼠肝脏缺口闭合酶使超螺旋DNA松弛的最佳一价阳离子浓度(Na+或K+)为150-200 mM。停止与碱的反应后,缺口DNA的检测取决于酶与DNA的高摩尔比,最大一价阳离子在50 - 100 mM之间。盐浓度在15 ~ 200mm范围内变化,对酶对缺口闭合反应的催化作用似乎没有影响。相反,在这两种试验中,不同的盐最佳值可以通过观察来解释,即在100毫米盐以下,缺口闭合酶通过过程机制起作用,而在150毫米盐以上,缺口闭合酶就变成非过程性的。从静止细胞染色质中对缺口闭合酶的盐洗脱似乎与从裸DNA中对酶的洗脱相似。然而,超过70%的染色质相关酶活性在300毫米盐下仍然与生长细胞的染色质结合,在这个浓度下,体外没有明显的与裸DNA结合。
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The effect of salt on the binding of the eucaryotic DNA nicking-closing enzyme to DNA and chromatin

The optimum monovalent cation concentration (Na+ or K+) for the relaxation of superhelical DNA by the rat liver nicking-closing enzyme under conditions of DNA excess was found to be 150–200 mM. The detection of a nicked DNA species after stopping a reaction with alkali depends on having a high molar ratio of enzyme to DNA and is maximal between 50 and 100 mM monovalen cation. Varying the salt concentration from 15 to 200 mM appears to have no effect on the catalysis of the nicking-closing reaction by the enzyme. Instead different salt optima in these two assays can be explained by the observation that the nicking-closing enzyme acts by a processive mechanism below 100 mM salt and becomes nonprocessive above 150 mM. The salt elution of the nicking-closing enzyme from resting cell chromatin appears to be similar to that which one would expect for the elution of the enzyme from naked DNA. However, greater than 70% of the chromatin associated enzyme activity remained bound to chromatin from growing cells at 300 mM salt, a concentration at which there is no significant binding to naked DNA in vitro.

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