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

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.