Biology subjects

Rossi, M. J.

Publications and source records attributed to Rossi, M. J..

3 recordsLinked to original sources

bioRxiv2025

RAD52 and RPA act in a concert promoting inverse RNA strand exchange

Recent studies in eukaryotes have revealed an important role of RNA in DNA repair and identified the RAD52 protein as a central player in RNA-dependent repair of DNA. In vitro, RAD52 promotes inverse RNA strand exchange between dsDNA and homologous RNA. This reaction is strongly stimulated by the RAD52 partner, replication protein A (RPA). Here, using NMR and biochemical methods we investigated the mechanism of this stimulation. We identified two RPA-binding sites in the unstructured RAD52 C-terminal domain (CTD), which mediate interaction with RPA70 and RPA32 subunits. These interactions are critical for stimulation of inverse RNA strand exchange. Furthermore, we showed that stimulation of inverse RNA strand exchange requires formation of an RPA-RNA complex that strengthens the RPA-RAD52 interaction and serves to deliver RNA to the RAD52-dsDNA complex for strand exchange. These results elucidate the mechanism of novel inverse RNA strand exchange activity of RAD52 and the role of RAD52-RPA interaction in RNA-dependent DNA repair.

biochemistry↗

bioRxiv2021

High similarity among ChEC-seq datasets

ChEC-seq is a method used to identify protein-DNA interactions across a genome. It involves fusing micrococcal nuclease (MNase) to a protein of interest. In principle, specific genome-wide interactions of the fusion protein with chromatin result in local DNA cleavages that can be mapped by DNA sequencing. ChEC-seq has been used to draw conclusions about broad gene-specificities of certain protein-DNA interactions. In particular, the transcriptional regulators SAGA, TFIID, and Mediator are reported to generally occupy the promoter/UAS of genes transcribed by RNA polymerase II in yeast. Here we compare published yeast ChEC-seq data performed with a variety of protein fusions across essentially all genes, and find high similarities with negative controls. We conclude that ChEC-seq patterning for SAGA, TFIID, and Mediator differ little from background at most promoter regions, and thus cannot be used to draw conclusions about broad gene specificity of these factors.

genomics↗

bioRxiv2019

Alignment and quantification of ChIP-exo crosslinking patterns reveal the spatial organization of protein-DNA complexes

The ChIP-exo assay precisely delineates protein-DNA crosslinking patterns by combining chromatin immunoprecipitation with 5' to 3' exonuclease digestion. Within a regulatory complex, the physical distance of a regulatory protein to DNA affects crosslinking efficiencies. Therefore, the spatial organization of a protein-DNA complex could potentially be inferred by analyzing how crosslinking signatures vary between the subunits of a regulatory complex. Here, we present a computational framework that aligns ChIP-exo crosslinking patterns from multiple proteins across a set of coordinately bound regulatory regions, and which detects and quantifies protein-DNA crosslinking events within the aligned profiles. By producing consistent measurements of protein-DNA crosslinking strengths across multiple proteins, our approach enables characterization of relative spatial organization within a regulatory complex. We demonstrate that our approach can recover aspects of regulatory complex spatial organization when applied to collections of ChIP-exo data that profile regulatory machinery at yeast ribosomal protein genes and yeast tRNA genes. We also demonstrate the ability to quantify changes in protein-DNA complex organization across conditions by applying our approach to data profiling Drosophila Pol II transcriptional components. Our results suggest that principled analyses of ChIP-exo crosslinking patterns enable inference of spatial organization within protein-DNA complexes.

bioinformatics↗