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by claude@2026-07, 2026-07-04
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The paper studied whether DNA polymerase alpha (Pol α), the replicative primase/polymerase, can bypass bulky DNA lesions via translesion synthesis, focusing specifically on cyclobutane pyrimidine dimers (CPDs). Using biochemical assays and single-molecule fluorescence/FRET approaches in vitro, the authors found that Pol α can replicate through CPD lesions, but that this intrinsic TLS activity is strongly inhibited by leading-strand replisome components, notably Pol ε and RPA, while Pol η showed the expected CPD TLS behavior that was unaffected by Pol ε and stimulated by RPA. Single-molecule FRET indicated that Pol α’s DNA binding cleft must remain open to accommodate a bulky CPD, consistent with relatively slow bypass kinetics. The authors explicitly frame these findings as intrinsic Pol α TLS being suppressed at the replication fork by the replisome during normal replication, though the work is based on in vitro reconstitution of polymerase and accessory factors. The paper does not explicitly discuss endometriosis or adenomyosis; it was included in the corpus via a keyword match in the upstream search index.
Abstract
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. RESEARCH HIGHLIGHTS Polymerase α has translesion synthesis (TLS) activity past a CPD lesion, the first demonstration of TLS activity at a bulky lesion by a replicative polymerase at physiological nucleotide levels. Intrinsic Pol α TLS activity is suppressed during normal replication by replisome components Pol ε and RPA. Pol ε suppresses TLS incorporation by Pol α by proofreading The binding cleft of Pol α stays open to accommodate a pyrimidine dimer. GRAPHICAL ABSTRACT Pol α harbors CPD TLS activity that is suppressed by RPA and Pol ε at the replication fork during normal replication.
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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.
RESEARCH HIGHLIGHTS
Polymerase α has translesion synthesis (TLS) activity past a CPD lesion, the first demonstration of TLS activity at a bulky lesion by a replicative polymerase at physiological nucleotide levels.
Intrinsic Pol α TLS activity is suppressed during normal replication by replisome components Pol ε and RPA.
Pol ε suppresses TLS incorporation by Pol α by proofreading
The binding cleft of Pol α stays open to accommodate a pyrimidine dimer.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
This version of the manuscript includes several new experiments including TLS kinetics data, assessing TLS inbhition by Pol epsilon proofreading, and rNTP incorporation, among other additions and changes to the manuscript flow.
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