STRUCTURAL INSIGHTS INTO A DNA POLYMERASE WITH NON-NATURAL REVERSE-TRANSCRIPTASE ACTIVITY

DNA polymerases are ubiquitous enzymes found in all kingdoms of life that replicate DNA using DNA-templates. Recent findings have shown that Geobacillus stearothermophilus DNA polymerase I large fragment (Bst), has the ability to reverse transcribe RNA (1) and an artificial genetic polymer known as threose nucleic acid, or TNA, into DNA (2). While DNA and RNA are structurally similar, TNA is distinct in (i) its use of the four-carbon threose instead of the five-carbon ribose as a sugar and consequently, (ii) its phosphodiester linkages where TNA is extended from a 3’-2’ direction. Hence, the structural characterization of Bst in complex with template/primer duplexes containing DNA, RNA, and TNA templates will shed light into the molecular mechanisms of Bst promiscuity. Herein, we propose to use single-crystal X-ray crystallography to determine the three-dimensional structure of Bst bound to an RNA/DNA primer/template duplex. Bst was overexpressed in E. coli cells and purified using tandem liquid column chromatography. Purified Bst was incubated with a 1.5 molar excess of the primer/template duplex and the resulting complex was used to set up approximately six hundred sparse-matrix crystallization conditions. Crystal growth was monitored periodically and layers of plate-like crystals were identified after three days from several conditions. Preliminary X-ray diffraction analyses of the crystals using an in-house X-ray source revealed weak diffraction (~6-8 Å). Further screening and optimization is underway to produce three dimensional, single crystals with better diffraction (3 Å or lower). Once diffraction quality crystals have been obtained, they will be used to determine an atomic resolution structure. The resulting Bst binary structure will be compared to previously solved binary structures of Bst with DNA and TNA templates with identical sequences.