9CI9

Crystal structure of human polymerase eta with incoming dCMPnPP nucleotide across threofuranosyl thymidine in DNA template at extension stage

Summary for 9CI9

• Entry DOI: 10.2210/pdb9ci9/pdb
• Descriptor: DNA polymerase eta, DNA (5'-D(*CP*AP*TP*GP*(TFT)P*TP*GP*AP*CP*GP*CP*T)-3'), DNA (5'-D(*AP*GP*CP*GP*TP*CP*AP*A)-3'), ... (7 entities in total)
• Functional Keywords: dna binding protein, dna polymerase, transferase, transferase-dna complex, transferase/dna
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 3
• Total formula weight: 55483.77

Authors

Tomar, R.,Stone, M.P.,Egli, M. (deposition date: 2024-07-02, release date: 2024-09-18, Last modification date: 2024-10-16)

Primary citation

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.
DNA Replication across alpha-l-(3'-2')-Threofuranosyl Nucleotides Mediated by Human DNA Polymerase eta.
Biochemistry, 63:2425-2439, 2024

PubMed Abstract: α-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 primer insertion and extension reactions catalyzed by human trans-lesion synthesis (TLS) DNA polymerase η (hPol η) opposite a TNA-modified template strand without and in combination with -alkyl thymine lesions. Across TNA-T (tT), hPol η inserted mostly dAMP and dGMP, dTMP and dCMP with lower efficiencies, followed by extension of the primer to a full-length product. hPol η inserted dAMP opposite -methyl and -ethyl analogs of tT, albeit with reduced efficiencies relative to tT. Crystal structures of ternary hPol η complexes with template tT and -methyl tT at the insertion and extension stages demonstrated that the shorter backbone and different connectivity of TNA compared to DNA (3' → 2' versus 5' → 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 Å on average from the α-phosphate of the incoming dNTP, consistent with insertion opposite and extension past the TNA residue by hPol η. Conversely, the crystal structure of a ternary hPol η·DNA·tTTP complex revealed that the primer's terminal 3'-OH was too distant from the tTTP α-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-à-vis unnatural nucleotides in the template and as the incoming nucleoside triphosphate.

PubMed: 39259676
DOI: 10.1021/acs.biochem.4c00387

Experimental method

X-RAY DIFFRACTION (2.09 Å)