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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 单分子真实的时间测序（也称为SMRTTM）是由Pacific BiosciencesTM开发的并行单分子DNA合成测序（1）。 该技术可应用于广泛的基因组学研究，包括从头基因组测序和个体全基因组测序，用于个性化医疗。 该技术的关键组成部分之一是具有长读取长度和稳定性的高性能DNA聚合酶。 通过使用组合和智能设计方法，我们已经获得了具有改进的读取长度、稳定性和保真度的突变酶。为了理解这种改进的机制，并为单分子研究制造更好的突变体，酶和酶/DNA复合物的高分辨率结构是必不可少的。 此外，这种复合物在测序反应下的光子暴露之前和之后的高分辨率结构将揭示聚合酶的光子损伤的一般机制，这仍然没有完全理解。 我们目前已经成功地结晶突变聚合酶和DNA复合物。以前的研究表明，这些晶体不？与旋转阳极X射线源（2）配合良好。 因此，我们建议在同步加速器上进行这项研究。 我们相信同步辐射对该项目的成功至关重要，该项目将提供对突变体如何在SMRTTM中工作的迫切需要的理解，并在改进个性化医疗工具方面产生重大影响。

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.