HIGH-RESOLUTION CRYSTAL STRUCTURES OF MUTANT DNA POLYMERASE WITH IMPROVED READ-L

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 单分子实时测序(SMRTTM)是太平洋生物科学(TM)(1)开发的一种基于合成的并行单分子DNA测序技术。这项技术可以应用于广泛的基因组研究，包括从头基因组测序和个性化医学的个体全基因组测序。这项技术的关键组成部分之一是具有长阅读长度和稳定性的高效DNA聚合酶。通过组合和智能设计方法，我们已经获得了具有更好的读数长度、稳定性和保真度的突变酶。为了了解这种改进的机制并为单分子研究创造更好的突变体，酶和酶/DNA复合体的高分辨率结构是必不可少的。此外，测序反应下这类复合体在光子暴露前后的高分辨结构将有助于揭示聚合酶的一般光子损伤机制，这一点目前仍不完全清楚。 我们目前已经成功地结晶了突变的聚合酶和DNA复合体。以前的研究表明，这些晶体在旋转的阳极X射线源(2)下不能很好地衍射。因此，我们建议在同步加速器进行这项研究。我们相信，同步辐射是这一项目成功的关键，这将为SMRTTM中突变体的工作方式提供亟需的了解，并对改进个性化药物的工具产生重大影响。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Single Molecule Real Time Sequencing (also known as SMRTTM) is a parallelized single molecule DNA sequencing by synthesis developed by Pacific BiosciencesTM (1). This technology can be applied for a broad range of genomics research including De novo genome sequencing and individual whole genome sequencing for personalized medicine. One of the critical components of the technology is high performing DNA polymerases with long read-length and stability. By using combinatorial and intelligent design approaches, we have obtained mutant enzymes with improved read-length, stability, and fidelity. To understand the mechanism of the improvement and to make even better mutants for single-molecule studies, high-resolution structures of the enzymes and the enzyme/DNA complexes are essential. In addition, high-resolution structures of such complexes before and after photon exposure under the sequencing reaction will shed light on the mechanism of photon damages of the polymerases in general, which is still not fully understood. We currently have successfully crystallized mutant polymerase and DNA complexes. Previous studies have shown that these crystals don?t diffract well with a rotating anode X-ray source (2). Therefore, we propose to do this study at synchrotron. We believe synchrotron radiation is critical in the success of this project that will provide much needed understanding of how the mutants work in SMRTTM and make a big impact in improving tools for personalized medicine.