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bioRxiv · 10.1101/2025.07.02.662766

DNA Polymerase α has pyrimidine dimer translesion activity that is suppressed during normal replication

Mukherjee, P.·Sarker, A.·Schauer, G.
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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 ), 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 . We observe that Pol {varepsilon}, the leading strand replicative polymerase, and RPA, a single-stranded DNA binding protein complex, both strongly inhibit CPD TLS activity of Pol . In contrast, Pol , the canonical TLS Pol for pyrimidine dimers, is unaffected by Pol {varepsilon} and is conversely stimulated by RPA. Finally, we demonstrate with single-molecule Fluorescence Resonance Energy Transfer (FRET) that the DNA binding cleft of Pol 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 is likely suppressed at the replication fork by the replisome itself during normal replication. RESEARCH HIGHLIGHTSO_LIPolymerase 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. C_LIO_LIIntrinsic Pol TLS activity is suppressed during normal replication by replisome components Pol {varepsilon} and RPA. C_LIO_LIPol {varepsilon} suppresses TLS incorporation by Pol by proofreading C_LIO_LIThe binding cleft of Pol stays open to accommodate a pyrimidine dimer. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/662766v2_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@1750e05org.highwire.dtl.DTLVardef@1eb8cdcorg.highwire.dtl.DTLVardef@16cdd39org.highwire.dtl.DTLVardef@2fd532_HPS_FORMAT_FIGEXP M_FIG Pol harbors CPD TLS activity that is suppressed by RPA and Pol {varepsilon} at the replication fork during normal replication. C_FIG

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Mukherjee, P., Sarker, A., Schauer, G.. 2025-07-04. DNA Polymerase α has pyrimidine dimer translesion activity that is suppressed during normal replication. https://doi.org/10.1101/2025.07.02.662766

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