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Updated: Apr 18, 2026
Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes
Published on: April 4, 2025
The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ
Kouji Hirota1, Kazunori Yoshikiyo2, Guillaume Guilbaud3
1Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-ku, Kyoto 606-8501, Japan Department of Chemistry, GraduateSchool of Science and Engineering, Tokyo Metropolitan University, Minami-Osawa, Hachioji- shi, Tokyo 192-0397, Japan.
Abstract:
The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with normal kinetics. Like its counterpart in yeast, POLD3 is required for fully effective TLS, its loss resulting in hypersensitivity to a variety of DNA damaging agents, a diminished ability to maintain replication fork progression after UV irradiation and a significant decrease in abasic site-induced mutagenesis in the immunoglobulin loci. However, these defects appear to be largely independent of Polζ, suggesting that POLD3 makes a significant contribution to TLS independently of Polζ in DT40 cells. Indeed, combining polη, polζ and pold3 mutations results in synthetic lethality. Additionally, we show in vitro that POLD3 promotes extension beyond an abasic by the Polδ holoenzyme suggesting that while POLD3 is not required for normal replication, it may help Polδ to complete abasic site bypass independently of canonical TLS polymerases.
Insights
The regulatory subunit POLD3 is crucial for DNA repair and mutagenesis, even outside its role in DNA polymerase ζ. DT40 cells lacking POLD3 show defects in translesion synthesis and DNA damage tolerance.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA polymerase δ (Polδ) is essential for DNA replication and repair.
- The regulatory subunit POLD3 plays a role in translesion synthesis (TLS) and mutagenesis in yeast.
- Chicken DT40 B lymphocytes offer a model to study DNA repair mechanisms.
Purpose of the Study:
- To investigate the function of POLD3 in chicken DT40 B lymphocytes.
- To determine POLD3's role in DNA repair, mutagenesis, and replication fork stability.
- To elucidate POLD3's contribution to translesion synthesis (TLS) independently of DNA polymerase ζ.
Main Methods:
- Gene knockout studies in DT40 B lymphocytes to create pold3-deficient cell lines.
- Assays for DNA damage sensitivity, UV-induced mutagenesis, and abasic site-induced mutagenesis.
- In vitro biochemical assays to assess Polδ holoenzyme activity.
- Analysis of synthetic lethality in cells with combined mutations in TLS polymerases and POLD3.
Main Results:
- DT40 cells deficient in POLD3 are viable and replicate undamaged DNA normally.
- POLD3-deficient cells exhibit hypersensitivity to DNA damaging agents and impaired replication fork progression after UV irradiation.
- A significant decrease in abasic site-induced mutagenesis was observed in pold3-deficient cells.
- POLD3's contribution to TLS and abasic site bypass appears largely independent of DNA polymerase ζ.
- Combined mutations in POLD3, polη, and polζ result in synthetic lethality.
- In vitro, POLD3 enhances Polδ holoenzyme extension beyond abasic sites.
Conclusions:
- POLD3 is essential for efficient translesion synthesis (TLS) and tolerance to DNA damage in DT40 cells, independent of its role in DNA polymerase ζ.
- POLD3 may facilitate abasic site bypass by the Polδ holoenzyme, suggesting a novel role in DNA repair.
- The synthetic lethality observed with polη, polζ, and pold3 mutations highlights the complex interplay of TLS polymerases in maintaining genome stability.
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