Schauer, G. | bioRxiv Science

bioRxiv2025

DNA Polymerase α has 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 ), 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

biochemistry↗

bioRxiv2019

Conscious perception of flickering stimuli in binocular rivalry and continuous flash suppression is not affected by tACS-induced SSR modulation

The content of conscious perception is known to correlate with steady-state responses (SSRs), yet their causal relationship remains unclear. Can we manipulate conscious perception by directly interfering with SSRs through transcranial alternating current stimulation (tACS)? Here, we directly addressed this question in three experiments involving binocular rivalry and continuous flash suppression (CFS). Specifically, while participants (N=24) viewed either binocular rivalry or tried to detect stimuli masked by CFS, we applied sham or real tACS across parieto-occipital cortex at either the same or a different frequency and phase as an SSR eliciting flicker stimulus. We found that tACS did not differentially affect conscious perception in the forms of predominance, CFS detection accuracy, reaction time, or metacognitive sensitivity, confirmed by Bayesian statistics. We conclude that tACS application at frequencies of stimulus-induced SSRs does not have perceptual effects and that SSRs may be epiphenomenal to conscious perception.

neuroscience↗

bioRxiv2018

Nuclease dead Cas9 is a programmable roadblock for DNA replication

DNA replication occurs on chromosomal DNA while processes such as DNA repair, recombination and transcription continue. However, we have limited experimental tools to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct fused to the photo-stable dL5 protein fluoromodule as a novel, targetable protein-DNA roadblock for studying replication fork arrest at the single-molecule level in vitro as well as in vivo. We find that the specifically bound dCas9-guideRNA complex arrests viral, bacterial and eukaryotic replication forks in vitro.

biochemistry↗