Structural basis for the selective incorporation of an artificial nucleotide opposite a DNA adduct by a DNA polymerase

Abstract

The possibility to sequence cytotoxic O-alkylG DNA adducts would greatly benefit research. Recently we reported a benzimidazole-derived nucleotide that is selectively incorporated opposite the damaged site by a mutated DNA polymerase. Here we provide the structural basis for this reaction which may spur future developments in DNA damage sequencing.

A gene-targeted polymerase-mediated strategy to identifyO6-methylguanine damage

“…This engineered polymerase is thought to increase favourable electrostatic interactions between the DNA backbone and the enzyme, thus promoting damage bypass. 25,26 For the primer extension reactions, KlenTaq M747K (20 nM), DNA (10 nM) and dNTPs (10 mM) were incubated at 55 1C for 10 min (Fig. S6, ESI †).…”

“…3A) showed BenziTP pairs opposite O 6 -MeG with two hydrogen bond interactions. 26 Starting from the ternary complex crystal structure (PDB ID: 3RTV) 26 with wild type KlenTaq, undamaged primer:template DNA, and an incoming dCTP we (1) removed the incoming dCTP (2) replaced the terminal 3 0 nucleotide on the primer strand with Benzi, and (3) inserted G and O 6 -MeG in the template (Fig. 3B) to reflect the configuration of the experiments described above.…”

Modified nucleic acids: replication, evolution, and next-generation therapeutics

“…This fidelity data foreshadows an important limitation in enzymatic XNA synthesis that will likely need to be addressed as the field moves forward. On the other hand, the promiscuous behavior of polymerases has been leveraged to read and record the presence of epigenetic DNA and RNA modifications via misincorporation "signatures" at the modifications [162][163][164][165][166][167][168][169][170]. In addition, the first instance of direct sequencing of an XNA was recently reported; FANA was sequenced using nanopore technology, albeit with relatively short read lengths [171].…”

Modified nucleoside triphosphates in bacterial research for in vitro and live-cell applications

“…4). 30,31 Walsh and Beuning reviewed a series of modified NTPs used as a means to investigate DNA polymerase specificity and to understand how DNA polymerases cope with non-natural NTPs in the context of mutagenesis and repair mechanisms. 32…”