Biology subjects

Roginskaya, V.

Publications and source records attributed to Roginskaya, V..

5 recordsLinked to original sources

bioRxiv2025

Nucleosome unwrapping and PARP1 allostery drive affinities for chromatin and DNA breaks

Poly[ADP-ribose] polymerase 1 (PARP1) detects DNA strand breaks that occur in duplex DNA and chromatin. We employed correlative optical tweezers and fluorescence microscopy to quantify how single molecules of PARP1 identify single-strand breaks (i.e., nicks), undamaged nucleosome core particles (NCP) and NCPs containing DNA nicks. Fluorescently-tagged PARP1 or PARP2 from nuclear extracts bound nicks with nanomolar affinity but did not engage undamaged dsDNA regions. In contrast, PARP1 avidly bound undamaged NCPs, and partial NCP unwrapping induced by DNA tension significantly increased the on rate and affinity. Catalytically dead PARP1 or EB-47 inhibition greatly increased PARP1 affinity to DNA nicks and undamaged NCP, implicating a mechanism where PARP1 reverse allostery regulates PARP1 retention to undamaged chromatin. We also monitored ADP-ribosylation in real time upon PARP1 binding undamaged or nicked NCPs. These results provide key mechanistic insights into domain allostery and how pharmacological intervention alters PARP1 binding dynamics for therapeutic impacts.

biophysics↗

bioRxiv2023

Single-molecule analysis of purified proteins and nuclear extracts: insights from 8-oxoguanine glycosylase 1

By observing one molecule at a time, single-molecule studies can offer detailed insights about biomolecular processes including on rates, off rates, and diffusivity of molecules on strands of DNA. A recent technological advance (Single-molecule Analysis of DNA-binding proteins from Nuclear Extracts, SMADNE) has lowered the barrier to entry for single-molecule studies, and single-molecule dynamics can now be determined directly out of nuclear extracts, providing information in an intermediate environment between purified proteins in isolation and the heterogeneity of a nucleus. To compare and contrast the single-molecule DNA binding dynamics in nuclear extracts versus purified proteins, combined optical tweezers and fluorescence microscopy experiments were performed with purified GFP-tagged 8-oxoguanine glycosylase 1 (OGG1), purified GFP-OGG1 spiked into nuclear extracts, and nuclear extracts from human cells overexpressing GFP-OGG1. We observed differences in undamaged DNA binding during DNA damage search in each of the three conditions. Purified GFP-OGG1 engaged undamaged DNA for a weighted average lifetime of 5.7 s and 21% of these events underwent DNA diffusion after binding. However, unlike other glycosylases studied by SMADNE, OGG1 does not bind non-damaged DNA efficiently in nuclear extracts. In contrast, GFP-OGG1 binding dynamics on DNA substrates containing oxidative damage were relatively similar in all three conditions, with the weighted average binding lifetimes varying from 2.2 s in nuclear extracts to 7.8 s with purified GFP-OGG1 in isolation. Finally, we compared the purified protein and nuclear extract approaches for a catalytically dead OGG1 variant (GFP-OGG1-K249Q). This variant greatly increased the binding lifetime for oxidative DNA damage, with the weighted average lifetime for GFP-OGG1-249Q in nuclear extracts at 15.4 s vs 10.7 s for the purified protein. SMADNE will provide a new window of observation into the behavior of nucleic acid binding proteins only accessible by biophysicists trained in protein purification and protein labeling.

biochemistry↗

bioRxiv2023

Single-molecule analysis reveals TDG exhibits multiple modes of linear diffusion to process 5-formylcytosine.

Base excision repair is the main pathway involved in active DNA demethylation. 5-formylctyosine and 5-carboxylcytosine, two oxidized moieties of methylated cytosine, are recognized and removed by thymine DNA glycosylase (TDG) to generate an abasic site. Using single molecule fluorescence experiments, we studied TDG in the presence and absence of 5-formylctyosine. TDG exhibits multiple modes of linear diffusion, including hopping and sliding, in search of a lesion. We probed TDG active site variants and truncated N-terminus revealing how these variants alter the lesion search and recognition mechanism of TDG. On DNA containing an undamaged nucleosome, TDG was found to either bypass, colocalize with, or encounter but not bypass the nucleosome. However, truncating the N-terminus reduced the number of interactions with the nucleosome. Our findings provide unprecedented mechanistic insights into how TDG searches for DNA lesions in chromatin.

biophysics↗

bioRxiv2022

Single-Molecule Analysis of DNA-binding proteins from Nuclear Extracts (SMADNE)

Single-molecule characterization of protein-DNA dynamics provides unprecedented mechanistic details about numerous nuclear processes. Here, we describe a new method that rapidly generates single-molecule information for fluorescently tagged proteins isolated from nuclear extracts of human cells. This approach determines binding lifetimes (koff), events per second ([~]kon), positional dependence (specificity), and characterizes 1D diffusion along DNA. We demonstrated the wide applicability of this approach on three forms of DNA damage using seven native DNA repair proteins and two structural variants, including: poly(ADP-ribose) polymerase (PARP1), heterodimeric ultraviolet-damaged DNA-binding protein (UV-DDB), xeroderma pigmentosum complementation group C protein (XPC), and 8-oxoguanine glycosylase 1 (OGG1). By measuring multiple fluorescent colors simultaneously, we additionally characterized the assembly and disassembly kinetics of multi-protein complexes on DNA. Thus, Single-Molecule Analysis of DNA-binding proteins from Nuclear Extracts (SMADNE) provides new insights about damage recognition and represents a universal technique that can be used to rapidly characterize numerous protein-DNA interactions.

molecular biology↗

bioRxiv2021

Telomeric 8-oxoguanine drives rapid premature senescence in the absence of telomere shortening

Oxidative stress is a primary cause of cellular senescence and contributes to the pathogenesis of numerous human diseases. Oxidative damage to telomeric DNA is proposed to trigger premature senescence by accelerating telomere shortening. Here we tested this model directly using a precision tool to produce the common base lesion 8-oxo-guanine (8oxoG) exclusively at telomeres in human fibroblast and epithelial cells. A single induction of telomeric 8oxoG is sufficient to trigger multiple hallmarks of p53-dependent senescence. Telomeric 8oxoG activates ATM and ATR signaling, and enriches for markers of telomere dysfunction in replicating, but not quiescent cells. Acute 8oxoG production fails to shorten telomeres, but rather generates fragile sites and delayed mitotic DNA synthesis at telomeres, indicative of impaired replication. Based on our results we propose that oxidative stress promotes rapid senescence by producing oxidative base lesions which drive replication-dependent telomere fragility and dysfunction in the absence of shortening and shelterin loss.

molecular biology↗