Louie D, Serwer P
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
J Mol Biol. 1994 Sep 30;242(4):547-58. doi: 10.1006/jmbi.1994.1599.
By steric exclusion of volume, neutral polymers both condense and increase the effective concentration of DNA random coils. Neutral polymers also stimulate the in vitro packaging of DNA in the capsids of some double-stranded DNA bacteriophages. In the present study, the physical effects of neutral polymers on DNA random coils have been quantified by assaying the DNA products at equilibrium of the following two reactions of the 12 nucleotide single-stranded complementary (cohesive) ends of mature bacteriophage lambda DNA: bimolecular joining of half-molecules of mature lambda DNA, and cyclization of intact lambda DNA; cyclization is used as a probe for unimolecular DNA condensation. The smaller neutral molecules, including polyethylene glycol of molecular mass 200 Da (PEG200), shift both reactions towards dissociation; this shift is partially correlated with reduced water activity. The larger PEGs (molecular mass of 1540 or more) shift both reactions towards association. Water activity-corrected equilibrium constants for the larger PEGs are found to increase as a function of PEG concentration. Below 2% to 3% (w/v) PEG, these equilibrium constants are independent of PEG molecular mass; at higher PEG concentrations, these equilibrium constants increase as the molecular mass of the PEG increases. The following conclusions are drawn. (1) Volume exclusion among PEG molecules is the primary cause of the PEG molecular mass-dependence of excluded volume. (2) At the lower PEG concentrations, the PEG radius obtained by quantification of excluded volume is usually equal to the hydrodynamic PEG radius. (3) At any given PEG concentration, the PEG-DNA excluded volume is approximately the same for bimolecular DNA joining as it is for unimolecular DNA cyclization. (4) Polymer-induced stimulation of in vitro bacteriophage DNA packaging is derived primarily from alteration of water activity, not alteration of excluded volume.
通过空间体积排阻，中性聚合物既能使DNA无规卷曲凝聚，又能提高其有效浓度。中性聚合物还能刺激某些双链DNA噬菌体衣壳中DNA的体外包装。在本研究中，通过测定成熟噬菌体λDNA的12个核苷酸单链互补（粘性）末端的以下两个反应平衡时的DNA产物，对中性聚合物对DNA无规卷曲的物理效应进行了定量：成熟λDNA半分子的双分子连接，以及完整λDNA的环化；环化用作单分子DNA凝聚的探针。较小的中性分子，包括分子量为200Da的聚乙二醇（PEG200），会使两个反应都向解离方向移动；这种移动与水活性降低部分相关。较大的聚乙二醇（分子量为1540或更大）会使两个反应都向缔合方向移动。发现较大聚乙二醇的水活性校正平衡常数随聚乙二醇浓度的增加而增加。在低于2%至3%（w/v）的聚乙二醇浓度下，这些平衡常数与聚乙二醇分子量无关；在较高的聚乙二醇浓度下，这些平衡常数随聚乙二醇分子量的增加而增加。得出以下结论。（1）聚乙二醇分子之间的体积排阻是排除体积对聚乙二醇分子量依赖性的主要原因。（2）在较低的聚乙二醇浓度下，通过排除体积定量得到的聚乙二醇半径通常等于流体动力学聚乙二醇半径。（3）在任何给定的聚乙二醇浓度下，双分子DNA连接的聚乙二醇-DNA排除体积与单分子DNA环化的聚乙二醇-DNA排除体积大致相同。（4）聚合物诱导的体外噬菌体DNA包装刺激主要源于水活性的改变，而非排除体积的改变。
