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Updated: Jan 23, 2026
Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes
Published on: April 4, 2025
DNA Polymerase α has pyrimidine dimer translesion activity that is suppressed during normal replication
Projit Mukherjee1, Abheerup Sarker1,2,3, Grant D Schauer1
1Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins CO 80523.
Abstract:
To ensure rapid and accurate DNA replication during S-phase, the cell uses DNA damage tolerance (DDT) pathways, leaving lesions to be repaired after completion of replication. One established DDT pathway is Translesion Synthesis (TLS), in which lesions are bypassed by specialized TLS Polymerases that work in conjunction with the replisome. Here we demonstrate that DNA Polymerase alpha (Pol α), the replicative primase/polymerase, can also unexpectedly replicate through bulky lesions in vitro. We use biochemical and single-molecule fluorescence assays to characterize cyclobutane pyrimidine dimer (CPD) TLS activity of Pol α. We observe that Pol ε, the leading strand replicative polymerase, and RPA, a single-stranded DNA binding protein complex, both strongly inhibit CPD TLS activity of Pol α. In contrast, Pol η, the canonical TLS Pol for pyrimidine dimers, is unaffected by Pol ε and is conversely stimulated by RPA. Finally, we demonstrate with single-molecule Fluorescence Resonance Energy Transfer (FRET) that the DNA binding cleft of Pol α must remain in the open state to accommodate a bulky CPD lesion during TLS, possibly accounting for the relatively slow kinetics of CPD bypass that we observe. The results suggest that the intrinsic bulky TLS activity of Pol α is likely suppressed at the replication fork by the replisome itself during normal replication.
Insights
DNA Polymerase alpha (Pol α) bypasses UV-induced pyrimidine dimers via Translesion Synthesis (TLS). However, its activity is suppressed by other replication proteins, suggesting regulation at the DNA replication fork.
Area of Science:
- Molecular Biology
- DNA Replication
- DNA Repair
Background:
- UV radiation damages DNA, forming pyrimidine dimers that stall replication forks.
- DNA damage tolerance (DDT) pathways, including Translesion Synthesis (TLS), allow replication to bypass lesions.
- Specialized TLS polymerases handle lesion bypass during DNA replication.
Purpose of the Study:
- To investigate the potential Translesion Synthesis (TLS) activity of DNA Polymerase alpha (Pol α) on cyclobutane pyrimidine dimer (CPD) lesions.
- To characterize the biochemical and genetic basis of Pol α's interaction with CPDs.
- To determine how other replication fork proteins affect Pol α's TLS activity.
Main Methods:
- Biochemical assays to assess Pol α's polymerase activity on CPD-containing DNA.
- Single-molecule Fluorescence Resonance Energy Transfer (FRET) to study DNA binding dynamics.
- Yeast genetics to evaluate the in vivo function of Pol α in UV sensitivity.
Main Results:
- DNA Polymerase alpha (Pol α) exhibits robust in vitro TLS activity on CPD lesions without misincorporation.
- Pol α's DNA binding cleft accommodates CPD lesions, suggesting a mechanism for bypass.
- Overexpression of Pol α did not rescue UV sensitivity in a TLS-deficient strain.
- Replication protein A (RPA), Pol ε, and Pol δ inhibit Pol α's CPD TLS activity.
Conclusions:
- DNA Polymerase alpha (Pol α) possesses intrinsic TLS activity capable of bypassing CPD lesions.
- The TLS activity of Pol α is actively suppressed by other replication fork components (RPA, Pol ε, Pol δ) during normal replication.
- This suppression mechanism likely prevents Pol α from engaging in TLS under physiological conditions, ensuring fidelity.
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