Single-molecule fluorescence imaging of DNA maintenance protein binding dynamics and activities on extended DNA.

Defining the molecular mechanisms by which genome maintenance proteins dynamically associate with and process DNA is essential to understand the potential avenues by which these stabilizing mechanisms are disrupted. Single-molecule fluorescence imaging (SMFI) of protein dynamics on extended DNA has greatly expanded our ability to accomplish this, as it captures singular biomolecular interactions - in all their complexity and diversity - without relying on ensemble-averaging of bulk protein activity as most traditional biochemical techniques must do. In this review, we discuss how SMFI studies with extended DNA have substantially contributed to genome stability research over the past two years.

High-speed AFM imaging reveals DNA capture and loop extrusion dynamics by cohesin-NIPBL.

3D chromatin organization plays a critical role in regulating gene expression, DNA replication, recombination, and repair. While initially discovered for its role in sister chromatid cohesion, emerging evidence suggests that the cohesin complex (SMC1, SMC3, RAD21, and SA1/SA2), facilitated by NIPBL, mediates topologically associating domains and chromatin loops through DNA loop extrusion. However, information on how conformational changes of cohesin-NIPBL drive its loading onto DNA, initiation, and growth of DNA loops is still lacking. In this study, high-speed atomic force microscopy imaging reveals that cohesin-NIPBL captures DNA through arm extension, assisted by feet (shorter protrusions), and followed by transfer of DNA to its lower compartment (SMC heads, RAD21, SA1, and NIPBL). While binding at the lower compartment, arm extension leads to the capture of a second DNA segment and the initiation of a DNA loop that is independent of ATP hydrolysis. The feet are likely contributed by the C-terminal domains of SA1 and NIPBL and can transiently bind to DNA to facilitate the loading of the cohesin complex onto DNA. Furthermore, high-speed atomic force microscopy imaging reveals distinct forward and reverse DNA loop extrusion steps by cohesin-NIPBL. These results advance our understanding of cohesin by establishing direct experimental evidence for a multistep DNA-binding mechanism mediated by dynamic protein conformational changes.

Single-molecule imaging of genome maintenance proteins encountering specific DNA sequences and structures.

DNA repair pathways are tightly regulated processes that recognize specific hallmarks of DNA damage and coordinate lesion repair through discrete mechanisms, all within the context of a three-dimensional chromatin landscape. Dysregulation or malfunction of any one of the protein constituents in these pathways can contribute to aging and a variety of diseases. While the collective action of these many proteins is what drives DNA repair on the organismal scale, it is the interactions between individual proteins and DNA that facilitate each step of these pathways. In much the same way that ensemble biochemical techniques have characterized the various steps of DNA repair pathways, single-molecule imaging (SMI) approaches zoom in further, characterizing the individual protein-DNA interactions that compose each pathway step. SMI techniques offer the high resolving power needed to characterize the molecular structure and functional dynamics of individual biological interactions on the nanoscale. In this review, we highlight how our lab has used SMI techniques - traditional atomic force microscopy (AFM) imaging in air, high-speed AFM (HS-AFM) in liquids, and the DNA tightrope assay - over the past decade to study protein-nucleic acid interactions involved in DNA repair, mitochondrial DNA replication, and telomere maintenance. We discuss how DNA substrates containing specific DNA sequences or structures that emulate DNA repair intermediates or telomeres were generated and validated. For each highlighted project, we discuss novel findings made possible by the spatial and temporal resolution offered by these SMI techniques and unique DNA substrates.

Densely methylated DNA traps Methyl-CpG-binding domain protein 2 but permits free diffusion by Methyl-CpG-binding domain protein 3.

The methyl-CpG-binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands). Therefore, in this work, we investigate the binding and diffusion of MBD2 and MBD3 on more biologically relevant DNA templates that contain a large CpG island or limited CpG sites. Using a combination of single-molecule and biophysical analyses, we show that both MBD2 and MBD3 diffuse freely and rapidly across unmethylated CpG-rich DNA. In contrast, we found methylation of large CpG islands traps MBD2 leading to stable and apparently static binding on the CpG island while MBD3 continues to diffuse freely. In addition, we demonstrate both proteins bend DNA, which is augmented by methylation. Together, these studies support a model in which MBD2-NuRD strongly localizes to and compacts methylated CpG islands while MBD3-NuRD can freely mobilize nucleosomes independent of methylation status.