Biology subjects

Flocco, M.

Publications and source records attributed to Flocco, M..

3 recordsLinked to original sources

bioRxiv2025

PARP1 inhibitors regulate PARP1 structure independent of DNA, reducing binding affinity for single strand breaks

AbstractCancers caused by mutations to the DNA repair machinery may be treated by inhibitors that target Poly(ADP-ribose) Polymerase 1 (PARP1). PARP inhibitors are thought to cause toxicity by trapping PARP1 at single strand breaks, preventing single strand break repair, thus leading to accumulation of DNA damage and cancer cell death. Intriguingly though, different PARP inhibitors display similar cellular toxicities and catalytic inhibition despite having widely varying trapping potencies. To better understand this apparent contradiction and identify complementary mechanisms of action, we here visualize the effect of inhibitors on individual PARP1 and PARP2 molecules by atomic force microscopy (AFM). We find, surprisingly, that inhibitors cause significant PARP1 compaction and loss of molecular flexibility also in the absence of DNA. This compaction correlates with the trapping potency of the inhibitor; and could be functionally relevant by reducing the subsequent binding of pre-treated PARP1 to DNA. Such changes are less pronounced for PARP2, which shares a high sequence identity with the PARP1 catalytic domain but lacks the DNA binding domain present in PARP1. Our findings reveal an additional, DNA-independent mechanism of action for PARP inhibitors, where PARP inhibitors with strong trapping potencies target PARP1 in the absence of DNA, compact their conformation and thereby reduce its ability to bind to DNA.

biophysics↗

bioRxiv2025

Co2+-mediated adsorption facilitates atomic force microscopy of DNA molecules at double-helix resolution

Atomic force microscopy (AFM) has demonstrated the ability to resolve single DNA molecules in liquid at a spatial resolution that is sufficient to visualize the double helix structure and variations therein. Such variations can be due to inherent configurational flexibility and may be related to, e.g., DNA sequence, ionic screening, supercoiling, or protein binding. These AFM experiments require DNA to be adhered to a solid and preferably flat support. For high-resolution, in-liquid AFM studies so far, such adhesion has commonly been achieved using Ni2+ ions to electrostatically bridge between the negatively charged DNA and a negatively charged, atomically flat mica surface, yet Ni2+ ions tend to cause precipitation of salts on the surface, increasing the risk of AFM tip contamination and increasing the corrugation of the support surface, making it harder to distinguish secondary DNA structure. Here, we report on a sample preparation protocol that, instead, relies on Co2+ ions to adhere DNA to mica. While the Co2+ is similarly effective as Ni2+ for facilitating DNA adsorption onto mica, it leads to significantly reduced salt precipitation with the potential to provide enhanced reproducibility in high-resolution DNA imaging by AFM. We expect this to substantially facilitate high-resolution AFM studies of DNA in aqueous solutions.

biophysics↗

bioRxiv2020

Single-molecule measurements reveal that PARP1 condenses DNA by loop formation

Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear enzyme that plays important roles in DNA repair, chromatin organization and transcription regulation. Although binding and activation of PARP1 by DNA damage sites has been extensively studied, little is known about how PARP1 binds to long stretches of undamaged DNA and how it could shape chromatin architecture. Here, using a combination of single-molecule techniques including magnetic tweezers and atomic force microscopy, we show that PARP1 binds and condenses undamaged, kilobase-length DNA subject to sub-picoNewton mechanical forces. Decondensation by high force proceeds through a series of discrete increases in extension, indicating that PARP1 stabilizes loops of DNA. This model is supported by DNA braiding experiments which show that PARP1 can bind at the intersection of two separate DNA molecules. PARP inhibitors do not affect the level of condensation of undamaged DNA, but act to block condensation reversal for damaged DNA in the presence of NAD+. Our findings establish a mechanism for PARP1 in the organization of chromatin structure.

biophysics↗