Crystal structures of DNA polymerase I capture novel intermediates in the DNA synthesis pathway

Abstract

High resolution crystal structures of DNA polymerase intermediates are needed to study the mechanism of DNA synthesis in cells. Here we report five crystal structures of DNA polymerase I that capture new conformations for the polymerase translocation and nucleotide pre-insertion steps in the DNA synthesis pathway. We suggest that these new structures, along with previously solved structures, highlight the dynamic nature of the finger subdomain in the enzyme active site.

In Vitro Selection of an ATP-Binding TNA Aptamer

“…Taq DNA polymerase and 10X ThermoPol buffer were purchased from New England Biolabs (NEB, Ipswich, MA, USA). Kod-RI TNA polymerase and Bst-LF DNA polymerase were expressed and purified as described previously [ 30 , 31 ]. TNA triphosphates bearing natural bases and 7-phenyl-7-deaza-guanine were synthesized as previously described [ 20 , 27 , 32 ].…”

Crystal structures of a natural DNA polymerase that functions as an XNA reverse transcriptase

“…deviation for Cα atoms: 0.4 and 0.7 for the FANA and TNA containing structures, respectively). Structural differences of this type indicate that the polymerase engages the chimeric P/T duplex in slightly different conformations than is normally observed for a natural DNA duplex (9).…”

“…As an initial step in this direction, we pursued crystal structures of the translocated product of Bst DNA polymerase, which capture the product of a single DNA nucleotide addition onto the 3′ end of the DNA primer. For these experiments, we used the same P/T duplex (Figure 2A) described in previous studies so that the resulting structures could be compared with earlier structures obtained for the all-natural DNA P/T system (9). The only exception was the FANA template, which contained uridine residues in place of thymidine residues, as FANA thymidine amidites are not commercially available.…”

“…We evaluated the enzyme active site of both structures to identify molecular interactions that would allow Bst DNA polymerase to reverse transcribe XNA templates back into DNA. Relative to the natural system (9), the O and O1 helices of the finger subdomain and a loop connecting strands β12 and β13 of the palm subdomain change orientation to compensate for differences in the helical geometry of the chimeric heteroduplexes (Figure 2B, C). Structural deviations along the amide backbone are more pronounced for the TNA bound structure than the FANA bound structure, which is consistent with FANA being a close structural analog of DNA (44).…”

“…Bst DNA polymerase, a member of the DNA polymerase I family, has been extensively studied as a model system for DNA synthesis (7,8). Static structures depicting the four key mechanistic steps of the replication cycle (translocation, substrate binding, pre-catalysis, and post-catalysis) and their associated conformational changes are well documented (9). Together, these structures expand our understanding of the mechanism of DNA synthesis by illuminating the reaction pathway.…”