Heath, G. R. | bioRxiv Science

bioRxiv2023

NanoLocz: Image analysis platform for AFM, high-speed AFM and localization AFM

NanoLocz is an open-source computer program designed for high-throughput automatic processing and single-particle analysis of Atomic Force Microscopy (AFM) image data. High-Speed AFM and Localization AFM (LAFM) enable the study of single molecules with increasingly higher spatiotemporal resolution. However, efficient and rapid analysis of the images and movies produced by these techniques can be challenging, often requiring the use of multiple image processing software applications and scripts. Here, we introduce NanoLocz, an AFM and high-speed AFM analysis program that facilitates various single-particle analysis workflows through a simple, interactive interface. Workflows include but are not limited to: single-particle tracking, single-particle topographic feature analysis, single-molecule LAFM, time-resolved LAFM, and simulation LAFM. The source code and installation instructions for NanoLocz are available at https://github.com/George-R-Heath/NanoLocz.

biophysics↗

bioRxiv2023

BRCA1-BARD1 combines multiple chromatin recognition modules to bridge nascent nucleosomes

Chromatin association of the BRCA1-BARD1 heterodimer is critical to promote homologous recombination repair of DNA double-strand breaks (DSBs) in S/G2. How the BRCA1-BARD1 complex interacts with chromatin that contains both damage induced histone H2A ubiquitin and inhibitory H4AK20 methylation is not fully understood. We characterised BRCA1-BARD1 binding and enzymatic activity to an array of mono- and di-nucleosome substrates using biochemical, structural, and single molecule imaging approaches. We find that the BRCA1-BARD1 complex preferentially interacts and modifies di-nucleosomes over mono-nucleosomes, allowing integration of H2A Lys-15 ubiquitylation signals with other chromatin modifications and features. Using high speed-AFM to provide real-time visualization of BRCA1-BARD1 complex recognising chromatin, we show a highly dynamic complex that bridges two nucleosomes and associates with the DNA linker region. Bridging is aided by multivalent cross-nucleosome interactions that enhance BRCA1-BARD1 E3 ubiqiutin ligase catalytic activity. Multivalent interactions across nucleosomes explains how BRCA1-BARD1 can recognize chromatin that retains partial di-methylation at H4 Lys-20 (H4K20me2), a parental histone mark that blocks BRCA1-BARD1 interaction with nucleosomes, to promote its enzymatic and DNA repair activities.

molecular biology↗