ENERGETICS OF DNA TWISTING .1. RELATION BETWEEN TWIST AND CYCLIZATION PROBABILITY
ENERGETICS OF DNA TWISTING .1. RELATION BETWEEN TWIST AND CYCLIZATION PROBABILITY
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DOI：
10.1016/s0022-2836(83)80198-3
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发表时间：
1983-01-01
影响因子：
5.6
通讯作者：
BALDWIN, RL
中科院分区：
文献类型：
--
作者：
SHORE, D;BALDWIN, RL
DNA的扭转势是通过测量EcoRI限制性片段的环化概率或j因子作为DNA扭转的函数来直接确定的。环化概率与限制片段内聚端环化的平衡常数Kc成正比。DNA的扭曲是通过对242 bp[碱基对]EcoRI限制性片段进行小的内部添加或删除来改变的。研究了12个DNA分子，长度从237-254 bp不等。环化概率由两种体系的噬菌体T4 DNA连接酶的共价闭合率来测量：一个线性限制片段与其环化形式处于平衡状态，半分子（由钝端核酸内切酶切割）与连接的半分子处于平衡状态。惊人的结果是，在这个DNA大小范围内，j因子在很大程度上取决于分数捻：总螺旋捻与最接近的整数之间的差。因此，j以振荡的方式依赖于DNA长度在237和254 bp之间，周期约为10 bp。这些数据给出了DNA扭曲的自由能作为扭曲的函数。j与DNA长度的曲线可以拟合为一个谐波扭转势，其扭转常数为C = 2.4倍。这个值与Barkley和Zimm（1979）对C的不同估计是合理一致的：C = 1.8倍。10-19 erg cm)，比插入DNA的乙尖的荧光去极化测量DNA扭转动力学得到的值略大（C = 1.4倍）。10- 19erg cm)或自旋标签研究。这些实验提供了扭转自由能的直接测量，并表明DNA扭转势是对称的。DNA螺旋在一个有缺口的圆圈中是连续的或几乎连续的；据推测，这是因为DNA堆叠相互作用维持了单链缺口上的双螺旋结构。其结果是，单个缺口DNA环的扭曲对小（simeq）来说是积分的。250 bp)的平面DNA环，在环化时扭曲发生变化。这一结论得到了支持，发现T4连接酶对单缺口环的关闭速率不依赖于DNA长度的大小变化，这与上面给出的线性限制片段环化的结果相反。对3种体系的连接酶关闭反应温度依赖性进行了测定：与环化分子平衡的线性限制片段；(2)单痕圈；(3)平衡的半分子和连接的半分子。温度依赖性数据证实，系统(1)和(3)处于平衡状态，通过连接酶闭合反应的速率来测量：这两个反应的速率随着温度的降低而强烈增加，这是对低温下连接内聚端比例增加的响应，而单个缺口环的闭合速率随着温度的升高而增加，这是对典型酶催化反应的预期。(1)和(2)的速率常数之比得到Kc，即环化的平衡常数。用这种方法得到的值与Mertz和Davis（1972）对Kc的EM测量结果相当一致。(3)和(2)的速率常数之比得到了半分子双分子结合的平衡常数Ka， Ka的温度依赖性得到了一个合理的值（-32(.+-)）。10) kcal/mol)表示EcoRI内聚端连接的焓。
The twisting potential of DNA was determined directly by a method that measures the cyclization probability or j-factor of EcoRI restriction fragments as a function of DNA twist. The cyclization probability is proportional to Kc, the equilibrium constant for cyclization of the restriction fragment via its cohesive end. The twist of the DNA is varied by making small internal additions to or deletions from a 242 bp [base pairs] EcoRI restriction fragment. A series of 12 DNA molecules was studied, which range in length from 237-254 bp. The cyclization probability is measured from the rates of covalent closure by phage T4 DNA ligase of 2 systems: a linear restriction fragment in equilibrium with its cyclized form and half molecules (cut by a blunt-end endonuclease) in equilibrium with joined half molecules. The striking result is that, in this DNA size range, the j-factor depends strongly on the fractional twist: the difference between the total helical twist and the nearest integer. Thus j depends in an oscillatory manner on DNA length between 237 and 254 bp with a period of about 10 bp. These data give the free energy of DNA twisting as a function of twist. The curve of j vs. DNA length can be fitted to a harmonic twisting potential with a torsional constant of C = 2.4 .times. 10-19 erg cm. This value is in reasonable agreement with different estimates of C made by Barkley and Zimm (1979: C = 1.8 .times. 10-19 erg cm) and is somewhat larger than the value obtained resulting from the kinetics of DNA twisting measured by fluorescence depolarization of ethidium intercalated into DNA (C = 1.4 .times. 10-19 erg cm) or from spin label studies. These experiments provide a direct measurement of the torsional free energy and they show that the DNA twisting potential is symmetric. The DNA helix is continuous or nearly so in a nicked circle; presumably this happens because the DNA stacking interaction maintains the double helix in register across a single-strand nick. As a consequence, the twist of a singly nicked DNA circle is integral for small (.simeq. 250 bp) planar DNA circles and there is a change in twist upon cyclization. This conclusion is supported by finding that the rate of closure by T4 ligase of singly nicked circles is not dependent on either small or large changes in DNA length, in contrast to the results given above for cyclization of linear restriction fragments. The temperature dependence of the ligase closure reaction was also measured for 3 systems: linear restriction fragments in equilibrium with cyclized molecules; (2) singly nicked circles; and (3) half molecules in equilibrium with joined half molecules. The temperature dependence data confirm that systems (1) and (3) are in equilibrium as measured by the rate of the ligase closure reaction: the rates of of these 2 reactions increase strongly with decreasing temperature, in response to the increasing fraction of joined cohesive ends at low temperatures, whereas the rate of closing singly nicked circles increases with increasing temperature, as expected for a typical enzyme-catalyzed reaction. The ratio of rate constants measured for (1) and (2) gives Kc, the equilibrium constant for cyclization. Values obtained in this way show reasonable agreement with EM measurement of Kc by Mertz and Davis (1972). The ratio of rate constants measured for (3) and (2) gives Ka, the equilibrium constant for bimolecular association of half molecules, and the temperature dependence of Ka gives a reasonable value (-32 (.+-. 10) kcal/mol) for the enthalpy of joining the EcoRI cohesive ends.