Structure and stability of Holliday junctions

描述（由申请人提供）：DNA霍利迪连接是重组、病毒整合和DNA修复中的重要结构中间体。 我们先前已经确定了一个四路霍尔戴结在一个反向重复序列的单晶结构。 [其他序列在同一晶体系统中结晶为标准B-DNA双链体]从这项研究中，d（ApCpC）三核苷酸被鉴定为该系统中稳定连接的核心。该序列基序的重要性已被本实验室最近对药物结合和甲基化连接的研究所证实。 目前的建议的目的是使用这种基本的晶体系统来表征序列和阳离子对DNA连接的结构和稳定性的影响。[In此外，我们建议确定该序列基序和在晶体系统中观察到的构象的细节是否也与溶液中的霍利迪结相关。长期目标是定义稳定和固定四向连接的因素，并了解这些连接如何作为遗传信息交换和DNA修复酶识别的位点。 建议进行研究，以确定所有的三核苷酸序列，结晶为霍利迪连接，因此，提供详细的结构信息的影响，这种重组中间体的序列。与此相关，我们将研究堆叠的双链臂的主要和次要凹槽中的取代基对这些结在该晶体系统中形成的能力的贡献。ACC-核心内的直接和溶剂介导的氢键相互作用对霍利迪连接形成的贡献将使用硫代磷酸酯键的DNA的晶体结构竞争测定来研究。 一组晶体学研究提出映射和表征的具体相互作用的路口与单价和二价金属离子，和多价阳离子精胺和亚精胺。 [提出了一系列凝胶电泳研究，以将晶体中观察到的序列和结构效应与溶液中的效应联系起来。 在第一组研究中，将检查连接的浓度依赖性形成。该试验将用于1）直接测定ACC-核心对连接形成的影响和2）通过体外进化过程筛选在溶液中稳定连接的所有三核苷酸序列。 在一组单独的实验中，将使用二维凝胶电泳测定来量化晶体中观察到的分子相互作用对在从负超螺旋质粒DNA挤出的十字形DNA的基部形成的四向连接的热力学稳定性的贡献。最后，一组的研究设计，以确定是否堆叠的臂之间的几何关系跨越交界处是依赖于序列或晶体相互作用。 从这些研究中，我们将定义相关的分子内和溶剂的相互作用，负责固定的位置和几何形状的四路霍利迪路口。

DESCRIPTION (provided by applicant): DNA Holliday junctions are important structural intermediates in recombination, viral integration and DNA repair. We had previously determined the single-crystal structure of a four-way HoIIiday junction in an inverted repeat sequence. [Other sequences crystallize as standard B-DNA duplexes in this same crystal system] From this study, a d(ApCpC) trinucleotide was identified as the core of the stable junction in this system The significance of this sequence motif has been confirmed by more recent studies on drug-bound and methylated junctions in this laboratory. The aim of the current proposal is to use this basic crystal system to characterize the effect of sequence and cations on the structure and stability of DNA junctions. [In addition, we propose to determine whether this sequence motif and the details of the conformation observed in the crystal system are also I relevant to Holliday junctions in solution.] The long range goal is to define the factors that stabilize and fix the four-way junction, and to understand how the junctions may serve as sites for the exchange of genetic information and for recognition by DNA repair enzymes. Studies are proposed to identify all trinucleotide sequences that crystaIIize as Holliday junctions and, therefore, provide detailed structural information on the effect of sequence on this recombination intermediate. Related to this, we will study the contribution of substituent groups in the major and minor grooves of the stacked duplex arms on the ability of these junctions to form in this crystal system. The contribution of the direct and solvent mediated hydrogen bonding interactions within the ACC-core on the formation of Holliday junctions will be studied using a crvstallographic competition assay on DNAs with phosphorothiate linkages. A set of crystallographic studies is proposed to map and characterize the specific interactions of junctions with monovalent and divalent metal ions, and the polyvalent cations spermine and spermidine. [A series of gel electrophoresis studies are proposed to relate the sequence and structural effects seen in the crystals with effects in solution. In the first set of studies, the concentration dependent formation of junctions will be examined. The assay will be used to 1) directly determine the effect of the ACC-core on the formation of junctions and 2) to screen though an in vitro evolution process alI trinucleotide sequences that stabilize junctions in solution.] In a separate set of experiments, a two-dimensional gel electrophoresis assay will be used to quantify the contributions of the molecular interactions seen in the crystals to the thermodynamic stability of four-way junctions formed at the base of cruciform DNAs extruded from negatively supercoiled plasmid DNAs. Finally, a set of studies are designed to determine whether the geometric relationship between the stacked arms across the junction is dependent on the sequence or on crystal interactions. From these studies, we will define the relevant intramolecular and solvent interactions that are responsible for fixing the location and geometry of four-way Holliday junctions.