8SCK

Bst DNA polymerase I Large Fragment mutant F710Y/D598A with 3'-amino primer, dGTP, and calcium time-resolved 4h

Summary for 8SCK

• Entry DOI: 10.2210/pdb8sck/pdb
• Descriptor: DNA polymerase I, DNA primer/product, DNA template, ... (8 entities in total)
• Functional Keywords: dna polymerase, np-dna, origin of life, time-resolved crystallography, dna binding protein, replication-dna complex, replication/dna
• Biological source: Geobacillus stearothermophilus; More
• Total number of polymer chains: 6
• Total formula weight: 147985.37

Authors

Fang, Z.,Lelyveld, V.S.,Szostak, J.W. (deposition date: 2023-04-05, release date: 2023-10-25, Last modification date: 2023-11-08)

Primary citation

Lelyveld, V.S.,Fang, Z.,Szostak, J.W.
Trivalent rare earth metal cofactors confer rapid NP-DNA polymerase activity.
Science, 382:423-429, 2023

PubMed Abstract: A DNA polymerase with a single mutation and a divalent calcium cofactor catalyzes the synthesis of unnatural N3'→P5' phosphoramidate (NP) bonds to form NP-DNA. However, this template-directed phosphoryl transfer activity remains orders of magnitude slower than native phosphodiester synthesis. Here, we used time-resolved x-ray crystallography to show that NP-DNA synthesis proceeds with a single detectable calcium ion in the active site. Using insights from isotopic and elemental effects, we propose that one-metal-ion electrophilic substrate activation is inferior to the native two-metal-ion mechanism. We found that this deficiency in divalent activation could be ameliorated by trivalent rare earth and post-transition metal cations, substantially enhancing NP-DNA synthesis. Scandium(III), in particular, confers highly specific NP activity with kinetics enhanced by more than 100-fold over calcium(II), yielding NP-DNA strands up to 100 nucleotides in length.

PubMed: 37883544
DOI: 10.1126/science.adh5339

Experimental method

X-RAY DIFFRACTION (2.3 Å)