Biology subjects

Hamdan, S. M.

Publications and source records attributed to Hamdan, S. M..

11 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

PAF15 stabilizes PCNA on DNA and slows down its sliding dynamics

The PCNA-associated factor 15 (PAF15) protein is essential for human genome stability by regulating DNA replication and repair through its interaction with proliferating cell nuclear antigen (PCNA). Despite its significance, the mechanistic details of the PAF15-PCNA interaction remain insufficiently understood. Here, we reveal how PAF15 influences the sliding dynamics and function of PCNA. Using single-molecule diffusion analysis on a DNA skybridge platform, we show that PAF15 binding stabilizes PCNA in its DNA-bound state, reduces its diffusion rate along the duplex, restricts accessibility to PCNA PIP-box binding sites, and regulates PCNA loading and unloading on DNA via replication factor C (RFC). Our results are consistent with its coordinating role in both high-fidelity replication and lesion bypass. These findings establish PAF15 as a key regulator of PCNA function, serving as a mobile platform for DNA-editing enzymes and thereby influencing genome maintenance.

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↗

bioRxiv2025

Real-time imaging of rotation during synthesis by the replisome

During chromosome replication, unwinding by the helicase and synthesis by the polymerases can lead to overwinding and supercoiling of DNA. The mechanical consequences of these events and resulting local dynamics at the replication fork are not well understood. To address these issues, we developed a transverse DNA flow-stretching approach to spatially resolve the parental, leading and lagging strands in real-time. Using bacteriophage T7 as a model system, this approach revealed bursts of high-speed replisome rotation that support continuous DNA synthesis. Surprisingly, excessive rotation does not reduce replisome speed, but increases pausing, reduces processivity, and increases the number of polymerases. Taken together, our observations reveal intrinsic pathways to overcome challenges posed by unfavorable DNA topologies during DNA replication.

biophysics↗

bioRxiv2024

Structure of the human CTF18 clamp loader bound to PCNA

Sliding clamps like PCNA are crucial processivity factors for replicative polymerases, requiring specific clamp loaders for loading onto DNA. The human alternative clamp loader CTF18-RFC interacts with the leading strand polymerase Pol {varepsilon} and loads PCNA onto primer/template DNA using its RFC pentameric module. Here, we provide a structural characterization of the human CTF18-RFC complex and its interaction with PCNA. Our cryo-EM data support that the Ctf8 and Dcc1 subunits of CTF18-RFC, which form the regulatory module interacting with Pol {varepsilon}, are flexibly tethered to the RFC module. A 2.9 [A] cryo-EM structure shows the RFC module bound to PCNA in an auto-inhibited conformation similar to the canonical RFC loader, marking the initial step of the clamp-loading reaction. The unique RFC1 (Ctf18) large subunit of CTF18-RFC, which based on the cryo-EM map shows high relative flexibility, is anchored to PCNA through an atypical low-affinity PIP box in the AAA+ domain and engages the RFC5 subunit using a novel {beta}-hairpin at the disordered N-terminus. We show that deletion of this {beta}-hairpin impairs the CTF18-RFC-PCNA complex stability, slows down clamp loading, and decreases the rate of primer synthesis by Pol {varepsilon}. Our research identifies distinctive structural characteristics of the human CTF18-RFC complex, providing insights into its role in PCNA loading and the stimulation of leading strand synthesis by Pol {varepsilon}.

biochemistry↗

bioRxiv2024

Internal unwinding mechanism of a replicative helicase

Ring-shaped hexameric helicases are nucleotide hydrolases that unwind double-stranded DNA into single strands, a necessary step in DNA replication. Two main questions are critical to understanding their function: the location of DNA strand separation within the helicase, and the precise dynamic linkage between nucleotide hydrolysis and DNA translocation. We explore these questions by employing cryo-EM to visualize the translocation mechanics of a AAA+ helicase, the SV40 Large Tumor Antigen (LTag), on forked DNA. We find the DNA fork nexus is positioned deep within the core helicase domain, with each DNA-binding loop from the six subunits securing the tracking strand by forming a paired staircase spiral that substitutes the passive strand. This structure forges an internal separation wedge, channeling the passive strand through a gap in the subunit C-tiers at the back of the helicase. Via cryo-EM continuous heterogeneity analysis we capture, and exhaustively model, a seamless spectrum of conformations of translocating LTag at high resolution. ATP hydrolysis at the tightest inter-subunit interface operates like an entropy switch, triggering coordinated rigid-body rotations of the subunit C-tiers and DNA-binding loops, resulting in directional escorting of the tracking strand through the central channel, concomitantly setting preparatory steps for cycle restart. Overall, we demonstrate a dynamic model for hydrolysis-coupled translocation and DNA unwinding by a model helicase active in replication, with implications for origin unwinding. High structural conservation of core helicase regions suggests this mechanism is applicable to hexameric helicases across domains.

biophysics↗

bioRxiv2024

Structure of Aquifex aeolicus Lumazine Synthase by Cryo-Electron Microscopy to 1.42 Angstrom Resolution

Single particle Cryo-Electron microscopy (Cryo-EM) has become an essential structural determination technique with recent hardware developments making it possible to reach atomic resolution at which individual atoms, including hydrogen atoms, can be resolved. Thus Cryo-EM allows not only unprecedented detail regarding the structural architecture of complexes but also a better understanding surrounding their chemical states. In this study we used the enzyme involved in the penultimate step of riboflavin biosynthesis as a test specimen to benchmark a recently installed microscope and determine if other protein complexes could reach a resolution of 1.5[A] or better which so far has only been achieved for the iron carrier ferritin. Using state of the art microscope and detector hardware as well as the latest software techniques to overcome microscope and sample limitations, a 1.42[A] map of Aquifex aeolicus lumazine synthase (AaLS) was obtained from a 48-hour microscope session. In addition to water molecules and ligands involved in AaLS function, we can observe positive density for [~]50% of hydrogen atoms. A small improvement in resolution was achieved by Ewald sphere correction which was expected to limit the resolution to [~]1.5[A] for a molecule of this diameter. Our study confirms that other protein complexes can be solved to near-atomic resolution. Future improvements in specimen preparation and protein complex stabilization may allow more flexible macromolecules to reach this level of resolution and should become a priority of study in the field.

biophysics↗

bioRxiv2022

Human HelQ and DNA polymerase δ interact to halt DNA synthesis and stimulate DNA single-strand annealing

DNA strand breaks can be repaired by base-pairing with unbroken homologous DNA, forming a template for new DNA synthesis that patches over the break site. In eukaryotes multiple DNA break repair pathways utilize DNA polymerase {delta} (Pol {delta}) to synthesise new DNA from the available 3OH at the strand break. Here we show that DNA synthesis by human Pol {delta} is halted by the HelQ DNA repair protein directly targeting isolated Pol {delta} or Pol {delta} in complex with PCNA and RPA. The mechanism is independent of DNA binding by HelQ or Pol {delta}, maps to a region of HelQ that also modulates RPA, and requires multiple Pol {delta} subunits. Interaction of HelQ with the POLD3 subunit of Pol {delta} stimulated DNA single-strand annealing activity of HelQ. The data implicates HelQ in preventing genetic instability by restraining DNA synthesis in multiple DNA break repair pathways.

biochemistry↗

bioRxiv2022

Rapid single-molecule characterisation of nucleic-acid enzymes

The activity of enzymes is traditionally characterised through bulk-phase biochemical methods that only report on population averages. Single-molecule methods are advantageous in elucidating kinetic and population heterogeneity but are often complicated, time consuming, and lacking statistical power. We present a highly generalisable and high-throughput single-molecule assay to rapidly characterise proteins involved in DNA metabolism. The assay exclusively relies on changes in total fluorescence intensity of surface-immobilised DNA templates as a result of DNA synthesis, unwinding or digestion. Combined with an automated data-analysis pipeline, our method provides enzymatic activity data of thousands of molecules in less than an hour. We demonstrate our method by characterising three fundamentally different nucleic-acid enzyme activities: digestion by the phage{lambda} exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe a previously unknown activity of the UvrD helicase to remove proteins tightly bound to the ends of DNA.

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↗