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

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.