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.

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.

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 N-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 N-furfuryl-dG or N-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 N-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.

4‐Cyanoindole‐2ʹ‐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‐2ʹ‐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‐2ʹ‐deoxyribonucleoside‐5ʹ‐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‐2ʹ‐deoxyribonucleoside (4CIN) and 4CIN phosphoramidite 4 Basic Protocol 2: Synthesis of 4‐cyanoindole‐ribonucleoside (4CINr) Basic Protocol 3: Synthesis of 4‐cyanoindole‐2ʹ‐deoxyribonucleoside‐5ʹ‐triphosphate (4CIN‐TP) Basic Protocol 4: Steady state incorporation kinetics of 2AP‐TP and 4CIN‐TP by a DNA polymerase

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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...

More than 100,000 lesions are generated on DNA per cell per day due to such agents as reactive oxygen species and ultraviolet radiation. Y‐family DNA polymerases are specialized to carry out translesion synthesis by replicating past the lesion and are conserved in all domains of life. There are two Y‐family polymerases in E. coli, DinB (Pol IV) and UmuD'2C (Pol V), as well as several in humans including Pol κ (hPol κ, a DinB ortholog). Both DinB and hPol κ are known to bypass minor groove adducts of guanine. Using primer extension assays on DNA substrates containing the minor groove adduct N2‐furfuryl‐dG as well as undamaged templates we found that loop 1 residues of DinB near the catalytic site are important for activity as well as the selection of the correct nucleotide. There is a similar trend in the loop 1 region of hPol κ. In single nucleotide incorporation assays, wild‐type and variants of both polymerases show a tendency to insert incorrect dT as well as the correct dC opposite the N2‐furfuryl‐dG adduct. This is also seen for wild‐type and variants of hPol κ on an undamaged DNA template. These observations suggest that the loop 1 region of both polymerases is important in correct nucleotide incorporation during translesion synthesis. This work is supported by the American Cancer Society.

Lone for taking the time to read my dissertation and provide comments. Additionally to Carla and Mary for participating in my committee meetings every year and giving me additional feedback and support.