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

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 to strongly inhibit Pol α CPD TLS activity, requiring exonuclease activity. We further see that RPA, a single-stranded DNA binding protein complex, strongly inhibits 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.