Characterizing the Structure and Function of E-DNA

Ho，PuiShingMCB-0090615本研究旨在表征胞嘧啶甲基化(D M5C)或溴化(D Br5C)诱导的一种新形式的DNA双螺旋的构象决定因素及其潜在意义。这种新构象是在d(GGCGm5CC)2和d(GGCGBr5CC)2的单晶结构中发现的。X射线结晶学发现的任何新的DNA构象产生的明显问题包括：1)其形成的序列要求是什么，2)这种形式是否存在于溶液中，以及3)结构是否相关？这个项目的长期目标是利用这些结构信息来定位E-DNA基因组序列，从而将其构象置于更广泛的生物学背景中。本项目的第一个目标是定义可以采用这种新构象的亲本序列d(GGCGm5CC)2之外的序列类型。第二个目标是通过定义一个光谱特征来确定E-DNA是否存在于溶液中，该特征将晶体中的构象与其在水溶液中的形成联系起来。第三个目标是检验这一假设，即E-DNA是通向A-DNA的途径中的离散中间体。研究将从序列基序d(GGCGCC)2的从B-DNA到A-DNA的完整晶体结构开始，并将这些结构作为分子模拟的基础，以绘制B-DNA到A-DNA转变的热力学图。这一结果将得到停流暂态动力学研究的证实。最终目的是验证这样的假设，即E-DNA的构象和溶剂结构促进脱氨反应，最终导致dm5C快速突变为dT核苷酸。将监测从同位素浓缩水中将18O掺入A-DNA、B-DNA和E-DNA晶体的情况。

Ho, PuiShingMCB-0090615This research is designed to characterize the conformational determinants and the potential significance of a new form of the DNA double-helix induced by cytosine methylation (d m5C) or bromination (d Br5C). This novel conformation was discovered in the single-crystal structure of d(GGCGm5CC)2 and d(GGCGBr5CC)2 . The obvious questions that arise with any new conformation of DNA discovered by X-ray crystallography include: 1) what are the sequence requirements for its formation, 2) does this form exist in solution, and 3) is the structure relevant? The long-term goal is to use this structural information to locate E-DNA ingenomic sequences and, consequently, place the conformation in a broader biological context.The first objective of this project is to define the types of sequences beyond the parent sequence d(GGCGm5CC)2 that can adopt this novel conformation. The second objective is to determine whether E-DNA exists in solution by defining a spectroscopic signature that links the conformation in the crystal with its formation in aqueous solution. The third objective is to test the hypothesis that E-DNA is a discrete intermediate in the pathway that leads to A-DNA. The studies will start with the complete set of crystal structures from B-DNA to A-DNA for the sequence motif d(GGCGCC)2 , and use these structures as a basis for molecular simulations to map the thermodynamics for the B-DNA to A-DNA transition. The results will be confirmed by stopped-flow transient kinetic studies. The final objective is to test the hypothesis that the conformation and solvent structure of E-DNA facilitates the deamination reaction that ultimately leads to the rapid mutation of dm5C to dT nucleotides. The incorporation of 18O from isotopically enriched water into the crystals of A-DNA, B-DNA, and E-DNAwill be monitored.