Structure and stability of Holliday junctions

描述(申请人提供)：DNA Holliday连接是重组、病毒整合和DNA修复的重要结构中间体。我们之前已经在反向重复序列中确定了四向HoIIiday结的单晶结构。[其他序列在同一晶体系统中结晶为标准的B-DNA双链]从本研究中，d(ApCpC)三核苷酸被确定为该系统中稳定连接的核心。该序列基序的意义已被本实验室最近对药物结合连接和甲基化连接的研究所证实。本提议的目的是利用这一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。[此外，我们建议确定该序列基序和在晶体体系中观察到的构象细节是否也与溶液中的Holliday连接有关。]长期目标是定义稳定和固定四向连接的因素，并了解连接如何作为遗传信息交换和DNA修复酶识别的位置。 有人建议进行研究，以鉴定所有结晶为Holliday连接的三核苷酸序列，从而提供关于序列对这种重组中间体影响的详细结构信息。与此相关，我们将研究堆积的双链臂的主槽和次槽中的取代基对这些结在该晶体体系中形成的能力的贡献。直接和溶剂介导的氢键相互作用在ACC核心内对Holliday连接的形成的贡献将通过对具有硫代磷键的DNA的晶格竞争分析来研究。提出了一系列的结晶学研究，以绘制和表征与单价和二价金属离子以及多价阳离子精胺和亚精胺的特定相互作用。 [建议进行一系列凝胶电泳法研究，将晶体中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，将考察浓度依赖的结的形成。该分析将用于1)直接确定ACC核心对连接形成的影响，以及2)通过体外进化过程筛选稳定溶液中连接的ALI三核苷酸序列。]在另一组实验中，二维凝胶电泳法将被用来量化晶体中看到的分子相互作用对由负超螺旋质粒DNA挤出的十字形DNA碱基形成的四向连接的热力学稳定性的贡献。最后，设计了一系列研究，以确定连接两端的堆叠臂之间的几何关系是取决于顺序还是取决于晶体相互作用。通过这些研究，我们将定义相关的分子内和溶剂相互作用，这些相互作用负责固定四向Holliday结的位置和几何形状。

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.