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Abstract Chromatin structure is dictated by nucleosome assembly and internucleosomal interactions. The tight wrapping of nucleosomes inhibits gene expression, but modifications to histone tails modulate chromatin structure, allowing for proper genetic function. The histone H4 tail is thought to play a large role in regulating chromatin structure. Here we investigated the structure of nucleosomes assembled with a tail-truncated H4 histone using Atomic Force Microscopy. We assembled tail-truncated H4 nucleosomes on DNA templates allowing for the assembly of mononucleosomes or dinucleosomes. Mononucleosomes assembled on nonspecific DNA led to decreased DNA wrapping efficiency. This effect is less pronounced for nucleosomes assembled on positioning motifs. Dinucleosome studies resulted in the discovery of two effects- truncation of the H4 tail does not diminish the preferential positioning observed in full-length nucleosomes, and internucleosomal interaction eliminates the DNA unwrapping effect. These findings provide insight on the role of histone H4 in chromatin structure and stability. 

Apurinic/apyrimidinic endonuclease 1 (APE1) is involved in DNA 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. Herein, we applied atomic force microscopy (AFM) to characterize the interactions of APE1 with DNA containing two well-separated G-rich segments. 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, and their yield was as high as 53%. Furthermore, APE1 is capable of binding two DNA segments leading to the formation of loops in the DNA–APE1 complexes. The analysis of looped APE1-DNA complexes revealed that APE1 can bridge G-rich segments of DNA. The yield of loops bridging two G-rich DNA segments was 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 led us to a model for the interaction of APE1 with DNA and the search for the specific sites. The implication of these new APE1 properties in organizing DNA, by bringing two distant sites together, for facilitating the scanning for damage and coordinating repair and transcription is discussed. 

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, an elevated stability in pre-synaptic complexes assembled with specific and non-specific DNA substrates was found. To explain these surprising observations, a theoretical approach that describes the assembly of these complexes and compares the predictions with the experiment was 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. 

The assembly of synaptic protein-DNA complexes by specialized proteins is critical for bringing together two distant sites within a DNA molecule or bridging two DNA molecules. The assembly of such synaptosomes is needed in numerous genetic processes requiring the interactions of two or more sites. The molecular mechanisms by which the protein brings the sites together, enabling the assembly of synaptosomes, remain unknown. Such proteins can utilize sliding, jumping, and segmental transfer pathways proposed for the single-site search process, but none of these pathways explains how the synaptosome assembles. Here we used restriction enzyme SfiI, that requires the assembly of synaptosome for DNA cleavage, as our experimental system and applied time-lapse, high-speed AFM to directly visualize the site search process accomplished by the SfiI enzyme. For the single-site SfiI-DNA complexes, we were able to directly visualize such pathways as sliding, jumping, and segmental site transfer. However, within the synaptic looped complexes, we visualized the threading and site-bound segment transfer as the synaptosome-specific search pathways for SfiI. In addition, we visualized sliding and jumping pathways for the loop dissociated complexes. Based on our data, we propose the site-search model for synaptic protein-DNA systems.