Three Human Pol ι Variants with Impaired Polymerase Activity Fail to Rescue H₂O₂ Sensitivity in POLI-Deficient Cells

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Human DNA polymerase ι (pol ι) plays a crucial role in DNA repair pathways, including translesion DNA synthesis (TLS) and base excision repair (BER).
• Genetic variations in pol ι can potentially alter its enzymatic functions and influence cellular responses to oxidative DNA damage.
• The functional consequences of specific pol ι missense germline variations on its dual activities and cellular protection remain largely uncharacterized.

Purpose of the Study:

• To investigate the biochemical and cellular effects of 10 human missense germline variations in DNA polymerase ι.
• To determine how these variations impact both the polymerase and deoxyribose phosphate (dRP) lyase activities of pol ι.
• To assess the functional significance of these variants in cellular resistance to oxidative genotoxic agents.

Main Methods:

• Recombinant human pol ι (residues 1-445) proteins were expressed and purified to assess polymerase and dRP lyase activities.
• Biochemical assays were employed to quantify the specificity constants (kcat/Km) for nucleotide insertion and dRP lyase efficiency.
• CRISPR/Cas9-mediated POLI knockout in HEK293 cells was used to create a cellular model for assessing oxidative stress sensitivity.
• Cell-based assays involving hydrogen peroxide (H2O2) exposure and complementation with wild-type or variant pol ι were performed.

Main Results:

• Three variants (K209Q, K228I, Q386R) exhibited significantly reduced dCTP insertion efficiency opposite G and 8-oxo-7,8-dihydroguanine.
• Five variants (R126C, K345E, R209Q, K228I, Q386R) displayed impaired dRP lyase activity compared to wild-type pol ι.
• POLI-deficient cells showed increased sensitivity to H2O2, which was rescued by wild-type pol ι.
• Variants R126C and K345E, despite having impaired dRP lyase activity but wild-type polymerase activity, fully rescued H2O2 sensitivity.
• Variants R209Q, K228I, and Q386R, impaired in both polymerase and dRP lyase activities, failed to rescue H2O2 sensitivity.

Conclusions:

• The study highlights the differential impact of pol ι variants on its polymerase and dRP lyase functions.
• Impaired polymerase activity, rather than impaired dRP lyase activity, is critical for pol ι's role in protecting cells against oxidative stress.
• Hypoactive pol ι variants may increase an individual's susceptibility to oxidative genotoxic agents, underscoring the importance of functional pol ι in DNA repair.