Biology subjects

Loparo, J.

Publications and source records attributed to Loparo, J..

6 recordsLinked to original sources

bioRxiv2026

ssDNA hand-off from RPA to Polθ drives microhomology-mediated end-joining

DNA polymerase theta-mediated end joining (TMEJ) is the primary mechanism of double strand break repair in mitosis and becomes essential in BRCA-deficient cells. RPA, the ssDNA-binding protein that coats resected DNA ends, has long been thought to inhibit this pathway by blocking the initial microhomology search in TMEJ. Here we find instead that RPA plays an essential activating role in TMEJ. Structural, biochemical, and genetic analyses support a model in which Pol{theta} binds directly to RPA at resected DNA ends and then competes with RPA for ssDNA. This releases ssDNA into the Pol{theta} helicase domain. We propose that threading two DNA ends through the Pol{theta} dimer in this manner initiates the search for microhomology. Together, our findings establish a detailed mechanism of how proteins can harness RPA to access ssDNA for critical genome maintenance transactions.

biochemistry↗

bioRxiv2026

A cell-free system for mitotically-stimulated TMEJ

Double-stranded DNA breaks (DSBs) are cytotoxic and must be repaired to avoid cell death or mutagenesis. DSBs that form in the S or G2 phases of the cell cycle undergo end resection, creating substrates for homologous recombination (HR) and alternative end joining pathways. Theta-mediated end joining (TMEJ) is a DNA polymerase {theta} (Pol {theta}) dependent repair pathway that joins resected DSB ends by annealing short regions of microhomology. TMEJ becomes particularly important when HR is compromised or during mitosis. Despite intensive investigation, the mechanism of TMEJ remains poorly understood, in part due to the lack of an efficient cell-free system that recapitulates this reaction. Here, we address this deficit using Xenopus egg extracts. We show that a linear DNA template containing a 3' single-stranded DNA overhang with terminal microhomology undergoes efficient intermolecular joining in nucleoplasmic extracts derived from Xenopus eggs. Joining is abolished upon Pol {theta} depletion, and restoration of joining requires Pol {theta} that is proficient in both ATPase and DNA polymerase activities. Furthermore, joining is accelerated by recombinant Cyclin B-CDK1, which also enhances the recruitment of Pol {theta} to single-stranded DNA ends. This assay provides a biochemically tractable approach for dissecting the mechanism and regulation of TMEJ in a physiologically complex environment.

molecular biology↗

bioRxiv2026

Chromosome topology gates productive RecA homology search

In homologous recombination, DNA repair depends on recombinase filaments finding homologous templates on chromosomes whose topology is continually remodeled by replication and transcription. How dynamic chromosome topology affects homology search and DNA repair remains unknown. By combining live-cell imaging of RecA-mediated homology search with single-molecule assays on topologically-defined DNA substrates, we show that DNA supercoiling acts as a selectivity filter for productive homology search. In Caulobacter crescentus cells, supercoiling is dispensable for the filament movement required for homology search, but required for homology target capture and repair. In vitro, filaments transiently sample both relaxed and supercoiled DNA, yet selectively commit to capture only on negatively supercoiled targets. Mechanistically, we find an extreme kinetic preference (~100-fold) of filaments towards negatively supercoiled DNA targets; this topological preference is independent of DNA compaction, pins plectonemes at the capture site, and is conserved across diverse bacterial RecA homologs. Our findings establish that chromosome topology physically regulates homology search and DNA repair.

biophysics↗

bioRxiv2025

Mechanisms of HSV-1 helicase-primase inhibition and replication fork complex assembly

Herpesviruses are widespread double-stranded DNA viruses that establish lifelong latency and cause various diseases. Although DNA polymerase-targeting antivirals are effective, increasing drug resistance underscores the need for alternatives. Helicase-primase inhibitors (HPIs) are promising antivirals, but their mechanisms of action are poorly defined. Furthermore, how the helicase-primase (H/P) complex and DNA polymerase coordinate genome replication is not well understood for herpesviruses. Here, we report cryo-EM structures of the herpes simplex virus (HSV) H/P complex bound to HPIs, showing that these lock the helicase-primase complex in an inactive conformation. Single-molecule assays reveal that HPIs cause helicase-primase complexes to pause in unwinding activity on DNA. The structure of an HPI-bound replication fork complex, comprising the H/P complex (UL5, UL52, and UL8) and polymerase holoenzyme (UL30 and UL42), reveals a previously uncharacterized interface bridging these complexes. These findings provide a structural framework for understanding herpesvirus replisome assembly and advancing inhibitor development.

microbiology↗

bioRxiv2022

Replication stalling activates SSB for recruitment of DNA damage tolerance factors

Translesion synthesis (TLS) polymerases bypass DNA lesions that block replicative polymerases, allowing cells to tolerate DNA damage encountered during replication. It is well known that most bacterial TLS polymerases must interact with the sliding clamp processivity factor to carry out TLS, but recent work in Escherichia coli has revealed that single-stranded DNA-binding protein (SSB) plays a key role in enriching the TLS polymerase Pol IV at stalled replication forks in the presence of DNA damage. It remains unclear how this interaction with SSB enriches Pol IV in a stalling-dependent manner given that SSB is always present at the replication fork. In this study we use single-molecule imaging in live E. coli cells to investigate this SSB-dependent enrichment of Pol IV. We find that Pol IV is enriched through its interaction with SSB in response to a range of different replication stresses, and that changes in SSB dynamics at stalled forks may explain this conditional Pol IV enrichment. Finally, we show that other SSB-interacting proteins (SIPs) are likewise selectively enriched in response to replication perturbations, suggesting that this mechanism is likely a general one for enrichment of repair factors near stalled replication forks.

biochemistry↗

bioRxiv2018

The sequence dependent search mechanism of EcoRI

One-dimensional search is an essential step in DNA target recognition. Theoretical studies have suggested that the sequence dependence of one-dimensional diffusion can help resolve the competing demands of fast search and high target affinity, a conflict known as the speed-selectivity paradox. The resolution requires that the diffusion energy landscape is correlated with the underlying specific binding energies. In this work, we report observations of one-dimensional search by QD labeled EcoRI. Our data supports the view that proteins search DNA via rotation coupled sliding over a corrugated energy landscape. We observed that while EcoRI primarily slides along DNA at low salt concentrations, at higher concentrations its diffusion is a combination of sliding and hopping. We also observed long-lived pauses at genomic star sites which differ by a single nucleotide from the target sequence. To reconcile these observations with prior biochemical and structural data, we propose a model of search in which the protein slides over a sequence independent energy landscape during fast search, but rapidly interconverts with a \"hemi-specific\" binding mode in which a half site is probed. This half site interaction stabilizes the transition to a fully specific mode of binding which can then lead to target recognition.

biophysics↗