Biology subjects

Schaich, M.

Publications and source records attributed to Schaich, M..

6 recordsLinked to original sources

bioRxiv2026

XRCC1 Enables the Efficient Local Search for DNA Damage by DNA Polymerase Beta

Oxidative DNA damage is a common threat to genomic integrity, arising from endogenous metabolic processes and environmental exposures. If unrepaired, such oxidative DNA damage promotes mutagenesis and genomic instability. Cells counter this through base excision repair (BER), a multi-step pathway requiring the coordinated action of several proteins. Central to BER, DNA polymerase beta (pol ) locates single-nucleotide (1-nt) gaps and inserts the correct nucleotide, while x-ray repair cross-complementing 1 (XRCC1) is a scaffold protein that forms a stable complex with pol to coordinate BER factors at DNA damage. XRCC1 enhances BER efficiency, though the mechanism by which this occurs is unclear. Pol {beta} is proposed to be recruited to DNA damage by undamaged DNA scanning interactions, but this behavior has not yet been directly observed. Additionally, the influence of other BER proteins on pol recruitment, particularly XRCC1, remains unclear. Here, we used correlative optical tweezers-fluorescence microscopy to visualize DNA search and damage recognition by pol and XRCC1. We characterize each factor individually, examine their behavior as the pol -XRCC1 complex, and assess their interplay with apurinic/apyrimidinic endonuclease 1 (APE1), the enzyme upstream of pol in BER. We find that pol locates damage through 3D-diffusion, whereas XRCC1 exhibits both 3D- and 1D-diffusion. In combination, XRCC1 dramatically shifts pol {beta} search towards 1D-diffusion, enabling interrogation of non-damaged DNA using both search mechanisms. When both APE1 and pol are present, the pol -1nt gap complex is highly stable, with APE1 largely unable to disrupt the damage-bound pol . Together, these findings demonstrate that XRCC1 reshapes pol {beta} search behavior to promote efficient local damage recognition, providing a mechanistic basis for how BER factors coordinate lesion detection and processing to maintain genomic stability.

biochemistry↗

bioRxiv2026

A Dynamic Search Mechanism Enables APE1 to Identify AP-Sites in DNA

Apurinic/apyrimidinic (AP) sites are among the most frequent DNA lesions, arising thousands of times per cell each day. These AP-sites threaten genomic stability and, if left unrepaired, can lead to mutagenesis and human disease. The essential base excision repair enzyme Apurinic/Apyrimidinic Endonuclease I (APE1) initiates repair by cleaving DNA at AP-sites, yet how APE1 efficiently locates and recognizes these lesions within vast excesses of undamaged DNA has remained poorly understood. Using single-molecule imaging, we show that APE1 employs a dynamic search strategy that integrates 1D and 3D diffusion to rapidly scan DNA. On non-damaged DNA, APE1 undergoes fast diffusion, enabling efficient interrogation of large genomic regions within a single binding event. Upon encountering an AP-site, APE1 transitions from a mobile search state into a stationary, lesion-bound complex that is retained at the site of damage. Additional experiments with APE1 variants demonstrate that the intrinsically disordered N-terminal domain of APE1 supports 1D diffusion, whereas residue R177 stabilizes APE1 at the AP-site once recognized, and the catalytic residues D210 and E96 facilitate enzyme release after cleavage. Together, these findings define the molecular basis by which APE1 balances rapid genome surveillance with stable lesion engagement and timely release. More broadly, this work provides a mechanistic framework for how DNA repair enzymes efficiently locate and process rare lesions embedded within an excess of undamaged DNA. Significance StatementDNA repair enzymes face the formidable challenge of locating rare lesions hidden within vast stretches of undamaged DNA. The essential enzyme APE1 is tasked with identifying and initiating repair of AP-sites, a frequent DNA lesion, to maintain genome integrity. We show that APE1 overcomes this challenge by using a dynamic search mechanism that integrates rapid scanning with precise lesion recognition. Its unstructured N-terminal domain facilitates fast movement along non-damaged DNA, while specific active site residues stabilize engagement at AP-sites and release after catalysis. This coordination of diffusion, recognition, and turnover allows APE1 to efficiently survey the genome and repair AP-sites to maintain genome stability.

biochemistry↗

bioRxiv2024

The human Shu complex promotes RAD51 activity by modulating RPA dynamics on ssDNA.

Templated DNA repair that occurs during homologous recombination and replication stress relies on RAD51. RAD51 activity is positively regulated by BRCA2 and the RAD51 paralogs. The Shu complex is a RAD51 paralog-containing complex consisting of SWSAP1 and SWS1. We demonstrate that SWSAP1-SWS1 binds RAD51, maintains RAD51 filament stability, and enables strand exchange. Using single molecule confocal fluorescence microscopy combined with optical tweezers, we show that SWSAP1-SWS1 decorates RAD51 filaments proficient for homologous recombination. We also find SWSAP1-SWS1 enhances RPA diffusion on ssDNA. Importantly, we show human sgSWSAP1 and sgSWS1 knockout cells are sensitive to pharmacological inhibition of PARP and APE1. Lastly, we identify cancer variants in SWSAP1 that alter SWS1 complex formation. Together, we show that SWSAP1-SWS1 stimulates RAD51-dependent high-fidelity repair and may be an important new cancer therapeutic target.

biochemistry↗

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↗

bioRxiv2019

Biophysical characterization reveals the similarities of liposomes produced using microfluidics and electroformation

Giant Unilamellar Vesicles (GUVs) are a versatile tool in many branches of science, including biophysics and synthetic biology. Octanol-Assisted Liposome Assembly (OLA), a recently developed microfluidic technique enables the production and testing of GUVs within a single device under highly controlled experimental conditions. It is therefore gaining significant interest as a platform for use in drug discovery, the production of artificial cells and more generally for controlled studies of the properties of lipid membranes. In this work, we expand the capabilities of the OLA technique by forming GUVs of tunable binary lipid mixtures of DOPC, DOPG and DOPE. Using fluorescence recovery after photobleaching we investigated the lateral diffusion coefficients of lipids in OLA liposomes and found the expected values in the range of 1 m2/s for the lipid systems tested. We studied the OLA derived GUVs under a range of conditions and compared the results with electroformed vesicles. Overall, we found the lateral diffusion coefficients of lipids in vesicles obtained with OLA to be quantitatively similar to those in vesicles obtained via traditional electroformation. Our results provide a quantitative biophysical validation of the quality of OLA derived GUVs, which will facilitate the wider use of this versatile platform.

biophysics↗

bioRxiv2019

An integrated microfluidic platform for quantifying drug permeation across biomimetic vesicle membranes

The low membrane permeability of candidate drug molecules is a major challenge in drug development and insufficient permeability is one reason for the failure of antibiotic treatment against bacteria. Quantifying drug transport across specific pathways in living systems is challenging since one typically lacks knowledge of the exact lipidome and proteome of the individual cells under investigation. Here, we quantify drug permeability across biomimetic liposome membranes, with comprehensive control over membrane composition. We integrate the microfluidic octanol-assisted liposome assembly platform with an optofluidic transport assay to create a complete microfluidic total analysis system for quantifying drug permeability. Our system enables us to form liposomes with charged lipids mimicking the negative charge of bacterial membranes at physiological salt and pH levels, which proved difficult with previous liposome formation techniques. Furthermore, the microfluidic technique yields an order of magnitude more liposomes per experiment than previous assays. We demonstrate the feasibility of the assay by determining the permeability coefficient of norfloxacin across biomimetic liposomes.

biophysics↗