Biology subjects

Raducanu, V.-S.

Publications and source records attributed to Raducanu, V.-S..

4 recordsLinked to original sources

bioRxiv2026

Structural basis for aphidicolin inhibition of human processive DNA synthesis

Eukaryotic DNA replication requires RNA--DNA primer synthesis by Pol and processive lagging- and leading-strand synthesis by Pol {delta} and Pol {epsilon} respectively, tethered to the PCNA clamp. Aphidicolin, a fungal diterpenoid, is a selective inhibitor of these B-family polymerases and a tool for inducing controlled replication stress, yet its structural mechanism has been defined only for the catalytic core of Pol . Here we report cryo-EM structures of human Pol {delta} and Pol {epsilon} holoenzymes bound to primer-template DNA, PCNA and aphidicolin at 3.0 [A] and 3.1 [A], respectively. In both, aphidicolin occupies the incoming dNTP pocket and flips the templating base outward, locking the fingers open and arresting synthesis while preserving the holoenzyme architecture. Aphidicolin occupies an architecturally conserved pocket in Pol , Pol {delta} and Pol {epsilon}, yet Pol {delta} is approximately 4-5-fold more sensitive than Pol {epsilon} in reconstituted PCNA-dependent assays. We attribute this to a grip beyond the templating base that anchors the template strand in Pol {delta} but engages it only loosely in Pol {epsilon}, allowing the strand to escape the aphidicolin trap. Selectivity therefore resides not in the inhibitor pocket but in template handling, defining a route to next-generation B-family polymerase inhibitors.

biophysics↗

bioRxiv2026

Structural basis of nick translation in human DNA replication

Nick translation during Okazaki fragment maturation requires iterative coordination of DNA polymerase delta (Pol {delta}), which displaces the downstream primer, and flap endonuclease FEN1, which cleaves the resulting flap, on the sliding clamp PCNA. The structural basis of this coordination is unknown. We present cryo-EM structures of human Pol {delta}-PCNA, Pol {delta}-PCNA-FEN1 and FEN1-PCNA on flap DNA, capturing four states of the nick translation cycle. Pol {delta} strand displacement emerges from structural elements intrinsic to the B-family fold rather than dedicated separation machinery, with a conserved palm loop acting as separation wedge and PCNA engagement required for melting of the downstream duplex. In the Pol {delta}-PCNA-FEN1 toolbelt, FEN1 is pre-positioned opposite Pol {delta} on PCNA to receive the substrate. Nucleotide removal triggers DNA handoff while both enzymes remain clamp-bound, followed by Pol {delta} dissociation. A post-handoff structure reveals stable DNA retention by FEN1-PCNA after flap cleavage, explaining the slow nick translation kinetics and the obligate role of Ligase 1 in sealing.

biophysics↗

bioRxiv2025

smDeepFLUOR: Single-Molecule Deep Learning Fluorescence Classification

Fluorescence intensity variation has long served as a primary readout for monitoring biological events. However, single-fluorophore signals arising from distinct molecular events often exhibit similar intensity profiles, making further classification challenging using conventional methods. In this study, we introduce smDeepFLUOR, a deep learning-based framework that resolves seemingly homogeneous spatiotemporal fluorescence signals by uncovering latent features imperceptible to conventional analyses. By leveraging a three-dimensional convolutional neural network trained on image sequences captured over 7 x 7 x 10 voxel windows, smDeepFLUOR reliably distinguishes specific from nonspecific protein binding, even across different experimental days, with an accuracy of up to 97%. Remarkably, smDeepFLUOR also captures real-time DNA synthesis kinetics by identifying subtle changes in the spatial distance between the fluorophore and the 3 end of nascent DNA, a feature undetectable by traditional methods. These classifications were achieved without incorporating explicit physical rules or engineered features, implying the presence of intrinsic, previously unrecognized differences in emission patterns. This approach significantly extends the analytical capabilities of single-molecule fluorescence imaging and opens new avenues for minimally labeled and label-free protein activities.

biophysics↗

bioRxiv2019

Structure of the processive human Pol δ holoenzyme

In eukaryotes, DNA polymerase {delta} (Pol {delta}) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol {delta}-DNA-PCNA complex in the absence and presence of FEN1. Pol {delta} is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol {delta}s activity in replicating the human genome.

biochemistry↗