6US5

DNA polymerase I Large Fragment from Bacillus stearothermophilus with DNA template, 3'-amino primer, dGpNHpp analog, and Mn2+

Summary for 6US5

• Entry DOI: 10.2210/pdb6us5/pdb
• Related: 6UR2 6UR4 6UR9
• Descriptor: DNA polymerase I, DNA (5'-D(*GP*CP*GP*AP*TP*CP*AP*GP*(C42))-3'), DNA (5'-D(*CP*AP*CP*GP*CP*TP*GP*AP*TP*CP*GP*CP*A)-3'), ... (7 entities in total)
• Functional Keywords: dna polymerase, 3'-amino terminal primer, gpnhpp, mn2+, replication-dna complex, replication/dna
• Biological source: Geobacillus stearothermophilus; More
• Total number of polymer chains: 6
• Total formula weight: 147295.01

Authors

Zhang, W.,Lelyveld, V.S.,Szostak, J.W. (deposition date: 2019-10-24, release date: 2020-03-18, Last modification date: 2023-10-11)

Primary citation

Lelyveld, V.S.,Zhang, W.,Szostak, J.W.
Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase.
Proc.Natl.Acad.Sci.USA, 117:7276-7283, 2020

PubMed Abstract: All known polymerases copy genetic material by catalyzing phosphodiester bond formation. This highly conserved activity proceeds by a common mechanism, such that incorporated nucleoside analogs terminate chain elongation if the resulting primer strand lacks a terminal hydroxyl group. Even conservatively substituted 3'-amino nucleotides generally act as chain terminators, and no enzymatic pathway for their polymerization has yet been found. Although 3'-amino nucleotides can be chemically coupled to yield stable oligonucleotides containing N3'→P5' phosphoramidate (NP) bonds, no such internucleotide linkages are known to occur in nature. Here, we report that 3'-amino terminated primers are, in fact, slowly extended by the DNA polymerase from in a template-directed manner. When its cofactor is Ca rather than Mg, the reaction is fivefold faster, permitting multiple turnover NP bond formation to yield NP-DNA strands from the corresponding 3'-amino-2',3'-dideoxynucleoside 5'-triphosphates. A single active site mutation further enhances the rate of NP-DNA synthesis by an additional 21-fold. We show that DNA-dependent NP-DNA polymerase activity depends on conserved active site residues and propose a likely mechanism for this activity based on a series of crystal structures of bound complexes. Our results significantly broaden the catalytic scope of polymerase activity and suggest the feasibility of a genetic transition between native nucleic acids and NP-DNA.

PubMed: 32188786
DOI: 10.1073/pnas.1922400117

Experimental method

X-RAY DIFFRACTION (2.25 Å)