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Characterization of Nine Cancer-Associated Variants in Human DNA Polymerase κ
Nicole M Antczak1, Alice R Walker2, Hannah R Stern1
1Department of Chemistry and Chemical Biology , Northeastern University , Boston , Massachusetts 02115 , United States.
Abstract:
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.
Insights
Cancer-associated DNA polymerase kappa (pol κ) variants show altered activity and stability. Some variants mimic wild-type pol κ, while others exhibit reduced function, impacting DNA damage bypass and potentially cancer progression.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- DNA damage-bypass Y-family DNA polymerases are crucial for maintaining genomic stability and tolerating DNA adducts, including those from chemotherapy.
- DNA polymerase kappa (pol κ), a human Y-family polymerase, specifically bypasses minor groove adducts and is inhibited by major groove adducts.
- Mutations in pol κ are linked to various cancers and influence chemotherapy response, highlighting its clinical relevance.
Purpose of the Study:
- To characterize the polymerization activity, DNA extension capabilities, stability, and dynamics of nine cancer-associated pol κ variants.
- To understand how single nucleotide polymorphisms affect pol κ function in the context of DNA damage and repair.
- To correlate in vitro enzymatic and stability data with molecular dynamics simulations to elucidate functional mechanisms.
Main Methods:
- Enzymatic assays were performed to measure polymerization activity on undamaged and damaged DNA for nine pol κ variants.
- The ability of variants to extend from mismatched or damaged primer termini was assessed.
- Thermal stability was evaluated, and molecular dynamics simulations were used to analyze protein-DNA interactions and conformational changes.
Main Results:
- Pol κ variants were categorized into three groups based on activity: similar to wild-type (WT), more active, and less active than WT.
- Reduced thermal stability was observed for variants R298H and Y432S.
- Molecular dynamics simulations suggested that active and inactive pol κ conformations are adopted depending on the protein variant and DNA adduct identity.
Conclusions:
- Cancer-associated pol κ variants exhibit diverse functional profiles, impacting their ability to bypass DNA damage.
- Specific variants display altered stability and conformational dynamics, potentially explaining their differential enzymatic activities.
- These findings provide insights into the role of pol κ variants in cancer development and chemotherapy response.
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