Biology subjects

Ghoneim, M.

Publications and source records attributed to Ghoneim, M..

4 recordsLinked to original sources

bioRxiv2025

Delineation of a novel assembly intermediate in retroviral integration pathway

Retroviral integration is mediated by viral integrase (IN) which synapses two viral long terminal repeat (LTR) DNA ends and produces a series of nucleoprotein complexes known as intasomes. While structural studies of mature intasomes have illuminated key aspects of their architecture and provided insights into the integration reaction, the sequence of events driving IN oligomerization and engagement of the viral DNA pairing remains unclear. Here, using complementary biochemical and biophysical approaches, including ensemble and single molecule FRET, we reveal that integration progresses through a key transient intermediate that leads to the mature intasome. We demonstrate that Rous Sarcoma Virus (RSV) intasome assembly pathway proceeds through a tetrameric intermediate where two IN dimers engage a single DNA end. This complex subsequently oligomerizes to form mature functional octameric intasome in which two DNA ends are juxtaposed for concerted integration. These findings provide mechanistic insights into the stepwise pathway of retroviral integration and define a previously uncharacterized intermediate critical for intasome maturation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/675266v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@57a82aorg.highwire.dtl.DTLVardef@e17110org.highwire.dtl.DTLVardef@c5f4c4org.highwire.dtl.DTLVardef@4886d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Rad52 sorts and stacks Rad51 at the DNA junction to promote homologous recombination

Homologous recombination (HR) repairs double-stranded DNA breaks (DSBs). The DSBs are resected to yield single-stranded DNA (ssDNA) that are coated by Replication Protein A (RPA). Rad51 is a recombinase and catalyzes strand invasion and the search for homology. However, it binds to ssDNA with lower affinity than RPA. Thus, mediator proteins such as Rad52/BRCA2 are required to promote Rad51 binding to RPA-coated ssDNA, but the underlying mechanisms remain poorly understood. Saccharomyces cerevisiae Rad52 interacts with Rad51 through two distinct binding modes. We here uncover that the Rad51-binding site in the disordered C-terminus of Rad52 (mode-1) sorts polydisperse Rad51 into discrete monomers. The second Rad51 binding site resides in the ordered N-terminal ring of Rad52 (mode-2), but this interaction occurs at only one position on the ring. In single molecule confocal fluorescence microscopy combined with optical tweezer analysis, we directly visualize filament formation using fluorescent-Rad51. Rad52 catalyzes Rad51 loading onto RPA-coated ssDNA, with a distinct preference for junctions, but no filament growth is observed. Deletion of the C-terminus of Rad52 results in loss of Rad51 sorting and abrogates Rad51 binding to RPA-coated DNA. While BRCA2 and Rad52 are structurally unrelated, many of these functional features are conserved. We describe a concerted Sort & Stack mechanism for mediator proteins in promoting HR.

biochemistry↗

bioRxiv2021

KERA: Analysis Tool for Multi-Process, Multi-State Single-Molecule Data

Molecular machines within cells dynamically assemble, disassemble, and reorganize. Molecular interactions between their components can be observed at the single-molecule level and quantified using colocalization single-molecule spectroscopy (CoSMoS), in which individual labeled molecules are seen transiently associating with a surface-tethered partner, or other total internal reflection fluorescence microscopy (TIRFM) approaches in which the interactions elicit changes in fluorescence in the labeled surface-tethered partner. When multiple interacting partners can form ternary, quaternary and higher order complexes, the types of spatial and temporal organization of these complexes can be deduced from the order of appearance and reorganization of the components. Time evolution of complex architectures can be followed by changes in the fluorescence behavior in multiple channels. Here, we describe the kinetic event resolving algorithm (KERA), a software tool for organizing and sorting the discretized fluorescent trajectories from a range of single-molecule experiments. KERA organizes the data in groups by transition patterns, and displays exhaustive dwell-time data for each interaction sequence. Enumerating and quantifying sequences of molecular interactions provides important information regarding the underlying mechanism of the assembly, dynamics and architecture of the macromolecular complexes. We demonstrate KERAs utility by analyzing conformational dynamics of two DNA binding proteins: RPA and XPD helicase.

biophysics↗

bioRxiv2019

Retroviral integration into nucleosomes throughDNA looping and sliding along the histone octamer

Retroviral integrase can efficiently utilise nucleosomes for insertion of the reverse-transcribed viral DNA. In face of the structural constraints imposed by the nucleosomal structure, integrase gains access to the scissile phosphodiester bonds by lifting DNA off the histone octamer at the site of integration. To clarify the mechanism of DNA looping by integrase, we determined a 3.9 [A] resolution structure of the prototype foamy virus intasome engaged with a nucleosome core particle. The structural data along with complementary single-molecule Forster resonance energy transfer measurements reveal twisting and sliding of the nucleosomal DNA arm proximal to the integration site. Sliding the nucleosomal DNA by approximately two base pairs along the histone octamer accommodates the necessary DNA lifting from the histone H2A-H2B subunits to allow engagement with the intasome. Thus, retroviral integration into nucleosomes involves the looping-and-sliding mechanism for nucleosomal DNA repositioning, bearing unexpected similarities with chromatin remodelers.

biochemistry↗