Characterizing the Structure and Function of E-DNA

Ho，PuiShingMCB-0090615本研究旨在表征由胞嘧啶甲基化（d m5 C）或溴化（d Br 5C）诱导的DNA双螺旋新形式的构象决定因素和潜在意义。在d（GGCGm 5CC）2和d（GGCGBr 5CC）2的单晶结构中发现了这种新的构象。X射线晶体学发现的任何新的DNA构象都会产生明显的问题，包括：1）它形成的序列要求是什么，2）这种形式存在于溶液中，3）结构相关吗？长期的目标是利用这些结构信息来定位E-DNA基因组序列，从而将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（GGCGm 5CC）2之外的序列类型。第二个目标是确定是否E-DNA存在于溶液中，通过定义一个光谱特征，连接在晶体中的构象与其在水溶液中的形成。第三个目标是检验E-DNA是导致A-DNA的途径中的离散中间体的假设。这些研究将从序列基序d（GGCGCC）2从B-DNA到A-DNA的完整晶体结构开始，并使用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。将通过停流瞬态动力学研究证实结果。最终的目的是测试的假设，构象和溶剂结构的E-DNA促进脱氨基反应，最终导致快速突变的dm 5C的dT核苷酸。将监测同位素富集水中18 O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.