Molecular Modeling of Unconventional DNA Polymerase Activities
Authors:Alison Fong, Melody Ho, Stephanie Kim, David Liu, Annie Surman, Sela Wang
- Angelika Niemz, Associate Professor, Keck Graduate Institute
- Deepali Shinde, Postdoctoral Fellow, Keck Graduate Institute
- Tatiana Prytkova, Assistant Professor, Chapman University
Nucleic acid amplification catalyzed by DNA polymerases is important in research and clinical diagnostics. For these applications, DNA polymerases have to effectively amplify specific targeted sequences, but should not cause non-specifically DNA amplification in the absence of target. In the isothermal EXPonential Amplification Reaction (EXPAR), we hypothesize based on prior work performed in our group that non-specific amplification may be caused by an unconventional polymerase activity, wherein the polymerase binds to a single stranded DNA template, and initiates DNA replication primed by a single dNTP bound in the enzyme’s active site. In prior work, we have identified certain sequence motifs in the DNA template that favor specific amplification, and other motifs that promote non-specific amplification. In this project, we used molecular modeling to further investigate this phenomenon. We docked single stranded EXPAR DNA template sequences (8 each with good and poor performance) into the active site of large fragment Bst DNA polymerase, starting from an X-ray structure available in the Protein Data Bank. We then performed molecular mechanics simulations using the HARLEM software suite to energy minimize these protein-DNA complex structures. Based on preliminary analysis of the resulting structures, we identified differences in the DNA backbone conformation for templates with good versus poor performance when bound to the polymerase active site. We further identified conformational changes of key amino acid residues that promote correct base pairing in the active site of the DNA polymerase. These results suggest that sequence motifs present in the DNA template can cause changes in the conformation of the polymerase-DNA complex, which may contribute to the likelihood of unconventional polymerase activities that lead to non-specific DNA amplification. However, further computational analysis plus experimental verification is required before definitive conclusions can be reached.