Characterization of Nine Cancer-Associated Variants in Human DNA Polymerase κ.

Specialized DNA damage-bypass Y-family DNA polymerases contribute to cancer prevention by providing cellular tolerance to DNA damage that can lead to mutations and contribute to cancer progression by increasing genomic instability. Y-family polymerases can also bypass DNA adducts caused by chemotherapy agents. One of the four human Y-family DNA polymerases, DNA polymerase (pol) κ, has been shown to be specific for bypass of minor groove adducts and inhibited by major groove adducts. In addition, mutations in the gene encoding pol κ are associated with different types of cancers as well as with chemotherapy responses. We characterized nine variants of pol κ whose identity was inferred from cancer-associated single nucleotide polymorphisms for polymerization activity on undamaged and damaged DNA, their abilities to extend from mismatched or damaged base pairs at primer termini, and overall stability and dynamics. We find that these pol κ variants generally fall into three categories: similar activity to wild-type (WT) pol κ (L21F, I39T, P169T, F192C, and E292K), more active than WT pol κ (S423R), and less active than pol κ (R219I, R298H, and Y432S). Of these, only pol κ variants R298H and Y432S had markedly reduced thermal stability. Molecular dynamics (MD) simulations with undamaged DNA revealed that the active variant F192C and more active variant S423R with either correct or incorrect incoming nucleotide mimic WT pol κ with the correct incoming nucleotide, whereas the less active variants R219I, R298H, and Y432S with the correct incoming nucleotide mimic WT pol κ with the incorrect incoming nucleotide. Thus, the observations from MD simulations suggest a possible explanation for the observed experimental results that pol κ adopts specific active and inactive conformations that depend on both the protein variant and the identity of the DNA adduct.

Human Y-Family DNA Polymerase κ Is More Tolerant to Changes in Its Active Site Loop than Its Ortholog Escherichia coli DinB.

DNA damage is a constant threat and can be bypassed in a process called translesion synthesis, which is typically carried out by Y-family DNA polymerases. Y-family DNA polymerases are conserved in all domains of life and tend to have specificity for certain types of DNA damage. Escherichia coli DinB and its human ortholog pol κ can bypass specific minor groove deoxyguanine adducts efficiently and are inhibited by major groove adducts, as Y-family DNA polymerases make contacts with the minor groove side of the DNA substrate and lack contacts with the major groove at the nascent base pair. DinB is inhibited by major groove adducts more than pol κ, and they each have active site loops of different lengths, with four additional amino acids in the DinB loop. We previously showed that the R35A active site loop mutation in DinB allows for bypass of the major groove adduct N6-furfuryl-dA. These observations led us to investigate the different active site loops by creating loop swap chimeras of DinB with a pol κ loop and vice versa by changing the loop residues in a stepwise fashion. We then determined their activity with undamaged DNA or DNA containing N2-furfuryl-dG or N6-furfuryl-dA. The DinB proteins with the pol kappa loop have low activity on all templates but have decreased misincorporation compared to either wild-type protein. The kappa proteins with the DinB loop retain activity on all templates and have decreased misincorporation compared to either wild-type protein. We assessed the thermal stability of the proteins and observed an increase in stability in the presence of all DNA templates and additional increases generally only in the presence of the undamaged and N2-furfuryl-dG adduct and dCTP, which correlates with activity. Overall we find that pol κ is more tolerant to changes in the active site loop than DinB.

Molecular beacons with oxidized bases report on substrate specificity of DNA oxoguanine glycosylases.

DNA glycosylase enzymes recognize and remove structurally distinct modified forms of DNA bases, thereby repairing genomic DNA from chemically induced damage or erasing epigenetic marks. However, these enzymes are often promiscuous, and advanced tools are needed to evaluate and engineer their substrate specificity. Thus, in the present study, we developed a new strategy to rapidly profile the substrate specificity of 8-oxoguanine glycosylases, which cleave biologically relevant oxidized forms of guanine. We monitored the enzymatic excision of fluorophore-labeled oligonucleotides containing synthetic modifications 8-oxoG and FapyG, or G. Using this molecular beacon approach, we identified several hOGG1 mutants with higher specificity for FapyG than 8-oxoG. This approach and the newly synthesized probes will be useful for the characterization of glycosylase substrate specificity and damage excision mechanisms, as well as for evaluating engineered enzymes with altered reactivities.

A Chemical Strategy for Intracellular Arming of an Endogenous Broad-Spectrum Antiviral Nucleotide.

The naturally occurring nucleotide 3'-deoxy-3',4'-didehydro-cytidine-5'-triphosphate (ddhCTP) was recently found to exert potent and broad-spectrum antiviral activity. However, nucleoside 5'-triphosphates in general are not cell-permeable, which precludes the direct use of ddhCTP as a therapeutic. To harness the therapeutic potential of this endogenous antiviral nucleotide, we synthesized phosphoramidate prodrug HLB-0532247 (1) and found it to result in dramatically elevated levels of ddhCTP in cells. We compared 1 and 3'-deoxy-3',4'-didehydro-cytidine (ddhC) and found that 1 more effectively reduces titers of Zika and West Nile viruses in cell culture with minimal nonspecific toxicity to host cells. We conclude that 1 is a promising antiviral agent based on a novel strategy of facilitating elevated levels of the endogenous ddhCTP antiviral nucleotide.

Synthesis of 4-Cyanoindole Nucleosides, 4-Cyanoindole-2'-Deoxyribonucleoside-5'-Triphosphate (4CIN-TP), and Enzymatic Incorporation of 4CIN-TP into DNA.

4-Cyanoindole-2'-deoxyribonucleoside (4CIN) is a fluorescent isomorphic nucleoside analogue with superior spectroscopic properties in terms of Stokes shift and quantum yield in comparison to the widely utilized isomorphic nucleoside analogue, 2-aminopurine-2'-deoxyribonucleoside (2APN). Notably, when inserted into single- or double-stranded DNA, 4CIN experiences substantially less in-strand fluorescence quenching compared to 2APN. Given the utility of these properties for a spectrum of research applications involving oligonucleotides and oligonucleotide-protein interactions (e.g., enzymatic processes, DNA hybridization, DNA damage), we envision that additional reagents based on 4-cyanoindole nucleosides may be widely utilized. This protocol expands on the previously published synthesis of 4CIN to include synthetic routes to both 4-cyanoindole-ribonucleoside (4CINr) and 4-cyanoindole-2'-deoxyribonucleoside-5'-triphosphate (4CIN-TP), as well as a method for the enzymatic incorporation of 4CIN-TP into DNA by a polymerase. These methods are anticipated to further enable the utilization of 4CIN in diverse applications involving DNA and RNA oligonucleotides. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of 4-cyanoindole-2'-deoxyribonucleoside (4CIN) and 4CIN phosphoramidite 4 Basic Protocol 2: Synthesis of 4-cyanoindole-ribonucleoside (4CINr) Basic Protocol 3: Synthesis of 4-cyanoindole-2'-deoxyribonucleoside-5'-triphosphate (4CIN-TP) Basic Protocol 4: Steady state incorporation kinetics of 2AP-TP and 4CIN-TP by a DNA polymerase.

Point mutations in Escherichia coli DNA pol V that confer resistance to non-cognate DNA damage also alter protein-protein interactions.

Y-family DNA polymerases are important for conferring cellular resistance to DNA damaging agents in part due to their specialized ability to copy damaged DNA. The Escherichia coli Y-family DNA polymerases are encoded by the umuDC and dinB genes. UmuC and the cleaved form of UmuD, UmuD', form UmuD'2C (pol V), which is able to bypass UV photoproducts such as cyclobutane pyrimidine dimers and 6-4 thymine-thymine dimers, whereas DinB is specialized to copy N(2)-dG adducts, such as N(2)-furfuryl-dG. To better understand this inherent specificity, we used hydroxylamine to generate a random library of UmuC variants from which we then selected those with the ability to confer survival to nitrofurazone (NFZ), which is believed to cause N(2)-furfuryl-dG lesions. We tested the ability of three of the selected UmuC variants, A9V, H282P, and T412I, to bypass N(2)-furfuryl-dG in vitro, and discovered that pol V containing UmuC A9V has overall modestly better primer extension activity than WT pol V, whereas the UmuC T412I and UmuC H282P mutations result in much lower primer extension efficiency. Upon further characterization, we found that the ability of the UmuC variant A9V to render cells UV-mutable is dependent on the proper length of the arm of UmuD'. Cells harboring UmuC variants T412I and H282P show enhanced cleavage of UmuD to form UmuD', which, together with our other observations, suggests that this may be due to a disruption of a direct interaction between UmuC and UmuD. Thus, we find that protein interactions as well as protein conformation appear to be crucial for resistance to specific types of DNA damage.