Download MendeleyDNA Replication across alpha-l-(3'-2')-Threofuranosyl Nucleotides Mediated by Human DNA Polymerase eta.
Tomar, R., Ghodke, P.P., Patra, A., Smyth, E., Pontarelli, A., Copp, W., Guengerich, F.P., Chaput, J.C., Wilds, C.J., Stone, M.P., Egli, M.(2024) Biochemistry 63: 2425-2439
- PubMed: 39259676
- DOI: https://doi.org/10.1021/acs.biochem.4c00387
- Primary Citation of Related Structures:
9CHW, 9CI9, 9CIH, 9CIQ, 9CJ9 - PubMed Abstract:
¦Á-l-(3'-2')-Threofuranosyl nucleic acid (TNA) pairs with itself, cross-pairs with DNA and RNA, and shows promise as a tool in synthetic genetics, diagnostics, and oligonucleotide therapeutics. We studied in vitro primer insertion and extension reactions catalyzed by human trans-lesion synthesis (TLS) DNA polymerase ¦Ç (hPol ¦Ç) opposite a TNA-modified template strand without and in combination with O 4 -alkyl thymine lesions. Across TNA-T (tT), hPol ¦Ç inserted mostly dAMP and dGMP, dTMP and dCMP with lower efficiencies, followed by extension of the primer to a full-length product. hPol ¦Ç inserted dAMP opposite O 4 -methyl and -ethyl analogs of tT, albeit with reduced efficiencies relative to tT. Crystal structures of ternary hPol ¦Ç complexes with template tT and O 4 -methyl tT at the insertion and extension stages demonstrated that the shorter backbone and different connectivity of TNA compared to DNA (3' ¡ú 2' versus 5' ¡ú 3', respectively) result in local differences in sugar orientations, adjacent phosphate spacings, and directions of glycosidic bonds. The 3'-OH of the primer's terminal thymine was positioned at 3.4 ? on average from the ¦Á-phosphate of the incoming dNTP, consistent with insertion opposite and extension past the TNA residue by hPol ¦Ç. Conversely, the crystal structure of a ternary hPol ¦Ç¡¤DNA¡¤tTTP complex revealed that the primer's terminal 3'-OH was too distant from the tTTP ¦Á-phosphate, consistent with the inability of the polymerase to incorporate TNA. Overall, our study provides a better understanding of the tolerance of a TLS DNA polymerase vis-¨¤-vis unnatural nucleotides in the template and as the incoming nucleoside triphosphate.
Organizational Affiliation:
Department of Chemistry, Vanderbilt Ingram Cancer Center, and Vanderbilt Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37235, United States.
