Structural Insights into DNA Replication without Hydrogen Bonds

Abstract

The genetic alphabet is comprised of two base pairs, and the development of a third, unnatural base pair would increase the genetic and chemical potential of DNA. d5SICS-dNaM is one of the most efficiently replicated unnatural base pairs identified to date, but its pairing is mediated by only hydrophobic and packing forces, and in free duplex DNA it forms a cross-strand intercalated structure that makes its efficient replication difficult to understand. Recent studies of the KlenTaq polymerase revealed that the insertion of d5SICSTP opposite dNaM proceeds via a mutually induced-fit mechanism, where the presence of the triphosphate induces the polymerase to form the catalytically competent closed structure, which in turn induces the pairing nucleotides of the developing unnatural base pair to adopt a planar Watson-Crick-like structure. To understand the remaining steps of replication, we now report the characterization of the pre-chemistry complexes corresponding to the insertion of dNaMTP opposite d5SICS, as well as multiple post-chemistry complexes in which the already formed unnatural base pair is positioned at the post-insertion site. Unlike with the insertion of d5SICSTP opposite dNaM, addition of dNaMTP does not fully induce the formation of the catalytically competent closed state. The data also reveal that once synthesized and translocated to the post-insertion position, the unnatural nucleobases again intercalate. Two modes of intercalation are observed, depending on the nature of the flanking nucleotides, and are each stabilized by different interactions with the polymerase, and each appear to reduce the affinity with which the next correct triphosphate binds. Thus, continued primer extension is limited by de-intercalation and rearrangements with the polymerase active site that are required to populate the catalytically active, triphosphate bound conformation.

The Expanded Genetic Alphabet

“…In contrast, addition of d NaM TP to a complex of KlenTaq bound to template containing d 5SICS does not fully induce the formation of the catalytically competent closed state. [167] However, it does induce the formation of a partially closed state, in which d 5SICS moves toward the insertion site and d NaM TP is bound to the O-helix via its triphosphate moiety. This state is similar to the ajar state, which is thought to allow the polymerase to test for correct base pairing during the synthesis of natural DNA.…”

“…[167] While structural differences were observed, in each case the UBP was positioned in the correct post-insertion site, where it again assumed an intercalated structure as it does in the free duplex.…”

“…Phosphates are indicated with open circles, natural nucleotides are indicated with open rectangles, and the unnatural nucleotides are indicated with grey and black boxes. Reproduced from Betz, et al [167] …”

A semisynthetic organism engineered for the stable expansion of the genetic alphabet

“…1A) being a particularly promising example (5-7). Despite lacking complementary hydrogen bonding, we demonstrated that the dNaM-d5SICS UBP is well replicated by a variety of DNA polymerases in vitro (7-10), and that this efficient replication is mediated by a unique mechanism that draws upon interbase hydrophobic and packing interactions (11,12). These efforts then culminated in the first progress toward the creation of an SSO in 2014, when we reported that Escherichia coli grown in the presence of the corresponding unnatural nucleoside triphosphates (dNaMTP and d5SICSTP), and provided with a plasmid-encoded nucleoside triphosphate transporter (NTT2) from Phaeodactylum tricornutum (which we denote as PtNTT2) (13), is able to import the unnatural triphosphates and replicate a single dNaM-d5SICS UBP on a second plasmid (14).…”

Recent progress in dissecting molecular recognition by DNA polymerases with non-native substrates

“…One of the most interesting discoveries identified an “ajar” BF conformation in the presence of a mismatched dNTP; the position of the active site’s O helix is intermediate between the “open” and “closed” states via a kink at residue G711 (Figure 1) [13]. Complexes with rare tautomers of incoming dNTPs [22], bulky dNTP analogs [14], and unnatural base pairs [15,16] also resulted in an “ajar” conformation. Taken together, these results indicate that various structures of polymerases can accommodate binding, but not catalysis, of some non-cognate substrates, and the resultant ajar conformations represent frozen states along the pathway for nucleotide discrimination.…”