Structures of a DNA Polymerase Inserting Therapeutic Nucleotide Analogues

Abstract

Members of the nucleoside analogue class of cancer therapeutics compete with canonical nucleotides to disrupt numerous cellular processes, including nucleotide homeostasis, DNA and RNA synthesis, and nucleotide metabolism. Nucleoside analogues are triphosphorylated and subsequently inserted into genomic DNA, contributing to the efficacy of therapeutic nucleosides in multiple ways. In some cases, the altered base acts as a mutagen, altering the DNA sequence to promote cellular death; in others, insertion of the altered nucleotide triggers DNA repair pathways, which produce lethal levels of cytotoxic intermediates such as single and double stranded DNA breaks. As a prerequisite to many of these biological outcomes, the modified nucleotide must be accommodated in the DNA polymerase active site during nucleotide insertion. Currently, the molecular contacts that mediate DNA polymerase insertion of modified nucleotides remain unknown for multiple therapeutic compounds, despite decades of clinical use. To determine how modified bases are inserted into duplex DNA, we used mammalian DNA polymerase β (pol β) to visualize the structural conformations of four therapeutically relevant modified nucleotides, 6-thio-2'-deoxyguanosine-5'-triphosphate (6-TdGTP), 5-fluoro-2'-deoxyuridine-5'-triphosphate (5-FdUTP), 5-formyl-deoxycytosine-5'-triphosphate (5-FodCTP), and 5-formyl-deoxyuridine-5'-triphosphate (5-FodUTP). Together, the structures reveal a pattern in which the modified nucleotides utilize Watson-Crick base pairing interactions similar to that of unmodified nucleotides. The nucleotide modifications were consistently positioned in the major groove of duplex DNA, accommodated by an open cavity in pol β. These results provide novel information for the rational design of new therapeutic nucleoside analogues and a greater understanding of how modified nucleotides are tolerated by polymerases.

A guardian residue hinders insertion of a Fapy•dGTP analog by modulating the open-closed DNA polymerase transition

“…In WT pol β, the formamide group clashes with the γ-oxygens and Cβ of Asp276 should the enzyme adopt the closed conformation (Figure 6B). To avoid this clash, the formamide would be forced into the major groove, similar to what was previously observed with other nucleotides containing modifications at the major groove C5 position (52). This transiently closed pol β conformation could be accommodated to a limited extent; however, the stabilizing contact with Asp276 and the templating base in the observed open pol β conformation must be preferred.…”

Mechanisms of telomerase inhibition by oxidized and therapeutic dNTPs

“…Third, 6-thio-dGTP has a very low IC 50 for telomerase repeat processivity inhibition. This is not surprising given that several human polymerases can insert 6-thio-dGTP with similar efficiencies as dGTP, and structures of Pol β inserting 6-thio-dGTP opposite C, versus dGTP, are nearly identical 56 , 57 . However, while 6-thio-dGTP can support DNA synthesis, it reduces primer elongation by DNA polymerases compared to dGTP 56 and strongly inhibits telomerase processivity.…”

“…While substitution of a 6-thio-dG for dG moderately decreases DNA duplex thermal stability, the opening rate of a 6-thio-dG:dC base pair is 17-fold faster than a dG:dC bp 59 . Structures indicate that the hydrogen bond is longer with the thio group and dC, compared to with the carbonyl group, in the Pol β active site 57 . Less is known about how 2-OH-dA impacts bp stability, however, alterations in the bp geometry with rU may impact the telomerase affinity for the RNA:DNA hybrid.…”

Structure and function relationships in mammalian DNA polymerases

“…Similar to other high fidelity polymerases, the fingers domain closes upon nucleotide binding to form a tight pocket around the inserted nucleotide to both position the dNTP for catalysis and reduce the frequency of mismatch insertions. Interestingly, this pol ε pocket includes a unique residue in the major groove (Tyr481), not conserved in pol δ, possibly contributing to its superior fidelity and potentially reducing the insertion efficiency of modified nucleotides that are handled efficiently by other DNA polymerases [85,86]. This structure also gave the first glimpse of an additional domain on pol ε not present in other members of the B-family, the P-domain (Figure 1A and D).…”