Trivalent rare earth metal cofactors confer rapid NP-DNA polymerase activity

• 成果类型: Article
• 署名作者: Lelyveld, Victor S.; Fang, Ziyuan; Szostak, Jack W.
• 署名单位: Harvard University; Harvard University Medical Affiliates; Massachusetts General Hospital; Harvard University; Harvard Medical School; University of Chicago; University of Chicago; Howard Hughes Medical Institute; Harvard University; Harvard University Medical Affiliates; Massachusetts General Hospital; Howard Hughes Medical Institute; Harvard University
• 刊物名称: SCIENCE
• ISSN/ISSBN: 0036-13281
• DOI: 10.1126/science.adh5339
• 发表日期: 2023-10-27
• 页码: 423-429
• 关键词: template-directed synthesis; transition-state; rna; FIDELITY; EVOLUTION; kinetics

摘要

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.