Vemulapalli, S. | bioRxiv Science

bioRxiv2024

Nanoscale interaction of endonuclease APE-1 with DNA characterized by atomic force microscopy

Apurinic/apyrimidinic endonuclease 1 (APE1) is involved in DNA replication, repair, and transcriptional regulation mechanisms. This multifunctional activity of APE1 should be supported by specific structural properties of APE1 that have not yet been elucidated. Here we applied atomic force microscopy (AFM) to characterize the interactions of APE1 with DNA. Complexes of APE1 with DNA containing G-rich segments were visualized, and analysis of the complexes revealed the affinity of APE1 to G-rich DNA sequences. Furthermore, loops in the DNA-APE1 complexes were visualized, and their yield was as high as 53 %. However, the loops were non-specific, with quantitative analysis revealing the yield of loops bridging two G-rich DNA segments to be 41%. Analysis of protein size in various complexes was performed, and these data showed that loops are formed by APE1 monomer, suggesting that APE1 has two DNA binding sites. The data lead us to a model for the interaction of APE1 with DNA that describes its molecular site search mechanism. The new properties of APE1 in organizing DNA, by bringing two distant sites together, may be important for facilitating the scanning for damage and coordinating repair and transcription.

biophysics↗

bioRxiv2023

Assembly of Synaptic Protein-DNA Complexes: Critical Role of Non-Specific Interactions

The synaptic protein-DNA complexes, formed by specialized proteins that bridge two or more distant sites on DNA, are critically involved in various genetic processes. However, the molecular mechanism by which the protein searches for these sites and how it brings them together is not well understood. Our previous studies directly visualized search pathways used by SfiI, and we identified two pathways, DNA threading and site-bound transfer pathways, specific to the site search process for synaptic DNA-protein systems. To investigate the molecular mechanism behind these site search pathways, we assembled complexes of SfiI with various DNA substrates corresponding to different transient states and measured their stability using a single-molecule fluorescence approach. These assemblies corresponded to specific-specific (synaptic), non-specific-non-specific (non-specific), and specific-non-specific (pre-synaptic) SfiI-DNA states. Unexpectedly, there was an elevated stability in pre-synaptic complexes assembled with specific and non-specific DNA substrates has been found. To explain these surprising observations, a theoretical approach that describes the assembly of these complexes and compares the predictions with the experiment is developed. The theory explains this effect by utilizing entropic arguments, according to which, after the partial dissociation, the non-specific DNA template has multiple possibilities of rebinding, effectively increasing the stability. Such difference in the stabilities of SfiI complexes with specific and non-specific DNA explains the utilization of threading and site-bound transfer pathways in the search process of synaptic protein-DNA complexes discovered in the time-lapse AFM experiments.

biophysics↗