Single-molecule force spectroscopy reveals binding and bridging dynamics of PARP1 and PARP2 at DNA double-strand breaks

Poly(ADP-ribose) polymerases (PARPs) play key roles in DNA damage repair pathways in eukaryotic cells. Human PARPs 1 and 2 are catalytically activated by damage in the form of both double-strand and single-strand DNA breaks. Recent structural work indicates that PARP2 can also bridge two DNA double-strand breaks (DSBs), revealing a potential role in stabilizing broken DNA ends. In this paper, we have developed a magnetic tweezers–based assay in order to measure the mechanical stability and interaction kinetics of proteins bridging across the two ends of a DNA DSB. We find that PARP2 forms a remarkably stable mechanical link (rupture force ~85 pN) across blunt-end 5′-phosphorylated DSBs and restores torsional continuity allowing DNA supercoiling. We characterize the rupture force for different overhang types and show that PARP2 switches between bridging and end-binding modes depending on whether the break is blunt-ended or has a short 5′ or 3′ overhang. In contrast, PARP1 was not observed to form a bridging interaction across blunt or short overhang DSBs and competed away PARP2 bridge formation, indicating that it binds stably but without linking together the two broken DNA ends. Our work gives insights into the fundamental mechanisms of PARP1 and PARP2 interactions at double-strand DNA breaks and presents a unique experimental approach to studying DNA DSB repair pathways

Combined single-molecule force and fluorescence measurements for biology

Recent advances in single-molecule techniques allow the application of force to an individual biomolecule whilst simultaneously monitoring its response using fluorescent probes. The effects of applied mechanical load on single-enzyme turnovers, biomolecular interactions and conformational changes can now be studied with nanometer precision and millisecond time resolution

Non-equilibrium mean-field theories on scale-free networks

Many non-equilibrium processes on scale-free networks present anomalous critical behavior that is not explained by standard mean-field theories. We propose a systematic method to derive stochastic equations for mean-field order parameters that implicitly account for the degree heterogeneity. The method is used to correctly predict the dynamical critical behavior of some binary spin models and reaction-diffusion processes. The validity of our non-equilibrium theory is furtherly supported by showing its relation with the generalized Landau theory of equilibrium critical phenomena on networks.Comment: 4 pages, no figures, major changes in the structure of the pape

Single-molecule force spectroscopy reveals binding and bridging dynamics of PARP1 and PARP2 at DNA double-strand breaks

Poly(ADP-ribose) polymerases (PARPs) play key roles in DNA damage repair pathways in eukaryotic cells. Human PARPs 1 and 2 are catalytically activated by damage in the form of both double-strand and single-strand DNA breaks. Recent structural work indicates that PARP2 can also bridge two DNA double-strand breaks (DSBs), revealing a potential role in stabilizing broken DNA ends. In this paper, we have developed a magnetic tweezers–based assay in order to measure the mechanical stability and interaction kinetics of proteins bridging across the two ends of a DNA DSB. We find that PARP2 forms a remarkably stable mechanical link (rupture force ~85 pN) across blunt-end 5′-phosphorylated DSBs and restores torsional continuity allowing DNA supercoiling. We characterize the rupture force for different overhang types and show that PARP2 switches between bridging and end-binding modes depending on whether the break is blunt-ended or has a short 5′ or 3′ overhang. In contrast, PARP1 was not observed to form a bridging interaction across blunt or short overhang DSBs and competed away PARP2 bridge formation, indicating that it binds stably but without linking together the two broken DNA ends. Our work gives insights into the fundamental mechanisms of PARP1 and PARP2 interactions at double-strand DNA breaks and presents a unique experimental approach to studying DNA DSB repair pathways

Heterogeneity of cell membrane structure studied by single molecule tracking

© The Royal Society of Chemistry 2021. his article is licensed under a Creative Commons Attribution 3.0 Unported Licence. https://creativecommons.org/licenses/by/3.0/Heterogeneity in cell membrane structure, typified by microdomains with different biophysical and biochemical properties, is thought to impact on a variety of cell functions. Integral membrane proteins act as nanometre-sized probes of the lipid environment and their thermally-driven movements can be used to report local variations in membrane properties. In the current study, we have used total internal reflection fluorescence microscopy (TIRFM) combined with super-resolution tracking of multiple individual molecules, in order to create high-resolution maps of local membrane viscosity. We used a quadrat sampling method and show how statistical tests for membrane heterogeneity can be conducted by analysing the paths of many molecules that pass through the same unit area of membrane. We describe experiments performed on cultured primary cells, stable cell lines and ex vivo tissue slices using a variety of membrane proteins, under different imaging conditions. In some cell types, we find no evidence for heterogeneity in mobility across the plasma membrane, but in others we find statistically significant differences with some regions of membrane showing significantly higher viscosity than others. This journal isPeer reviewedFinal Published versio

Monomeric PcrA helicase processively unwinds plasmid lengths of DNA in the presence of the initiator protein RepD

The helicase PcrA unwinds DNA during asymmetric replication of plasmids, acting with an initiator protein, in our case RepD. Detailed kinetics of PcrA activity were measured using bulk solution and a single-molecule imaging technique to investigate the oligomeric state of the active helicase complex, its processivity and the mechanism of unwinding. By tethering either DNA or PcrA to a microscope coverslip surface, unwinding of both linear and natural circular plasmid DNA by PcrA/RepD was followed in real-time using total internal reflection fluorescence microscopy. Visualization was achieved using a fluorescent single-stranded DNA-binding protein. The single-molecule data show that PcrA, in combination with RepD, can unwind plasmid lengths of DNA in a single run, and that PcrA is active as a monomer. Although the average rate of unwinding was similar in single-molecule and bulk solution assays, the single-molecule experiments revealed a wide distribution of unwinding speeds by different molecules. The average rate of unwinding was several-fold slower than the PcrA translocation rate on single-stranded DNA, suggesting that DNA unwinding may proceed via a partially passive mechanism. However, the fastest dsDNA unwinding rates measured in the single-molecule unwinding assays approached the PcrA translocation speed measured on ssDNA

Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility

Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain MTIP, and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC) and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion

Myosin-5, kinesin-1 and myosin-17 cooperate in secretion of fungal chitin synthase

The interactions between cytoskeletal motors are here explored in the context of polarized secretion in fungi, revealing unexpected complexity in the regulation of vesicle transport to and docking at the hyphal tip