DNA dynamics and single-molecule biology.

DNA stores information. Its function as a universal genetic material is among the most highly conserved qualities of living things. Its system of four bases, when overlaid with spatial and temporal controls, governs biology across the entire scale of life, from enzymatic reactions inside E. coli to embryogenesis in humans. Because the primary function of DNA is to store and propagate information, its sequence is often taken to be its most important biochemical property. However, during the ordinary course of cellular activity, DNA must be manipulated in many ways as a physical structure—a polymer in solution—regardless of whether a specific sequence is relevant to a given process. The dominant functional unit of this manipulation in the cell is the interaction between DNA and protein. Some interactions access or even extract the underlying sequence content of the DNA, whereas many others are largely oblivious to that information. DNA’s primary function in storing genetic information requires a degree of stability. Indeed, the cell’s ability to correctly maintain and propagate DNA sequences through many cycles of duplication at low error rates is the source of both the continuity and the variability so fundamental to evolution.1 DNA is also constrained by the more immediate and practical concerns of the cell, which, in handling and passing on genetic content, must efficiently carry out duplication of chromosomes.2 The two intertwined factors of long-term stability and duplication efficiency are served by the fairly uniform global structure of DNA. The familiar double helix is generally not taken to vary significantly in structure along its length, and this is true on average—for example, across the span of a large genomic fragment. However, the physical properties of DNA are not uniform across regions of a size relevant to protein binding.3 Nor are they uniform across cellular environments, which are heterogeneous and fluctuating,2a,4 or even across experimental conditions.5 Appreciating the basic physical qualities of DNA as a function of physiologically and experimentally relevant variables is essential to understanding protein–DNA interactions, and the physical properties of DNA are particularly important when interpreting the results of single-molecule experiments. Important factors to consider are the polymer nature of DNA and the effect of various forces on this polymer, the effect of temperature and salt concentration, DNA length, and sequence-dependent variations in structure. Furthermore, DNA dynamics influence complex, subtle, and time-dependent protein behaviors on short spatial and temporal scales, a swath of which are only accessible through single-molecule methodologies. (Recent reviews that explore the nature of single-molecule experiments include refs (6a−6c)). In this review, we focus on illustrative examples from our laboratory that have employed “DNA curtains”: parallel arrays of individual DNA molecules used to visualize the behavior of DNA-interacting proteins. However, we consider these concepts broadly applicable to single-molecule studies. Single-molecule bioscience is still coming of age, and given recent technical and conceptual advances, it is poised to tackle progressively more complex and physiologically relevant problems. However, designing and interpreting single-molecule experiments to study protein–DNA interactions often does not map directly onto comparable biochemical studies, and yet characterizing a particular system comprehensively requires data from as many approaches as possible. Constructing such cohesive understanding from disparate viewpoints, all directed toward an understanding of the same biological system, requires that each field possess a general framework to interpret results which can be universally applied to interpretation of phenomena as witnessed by that field. A successful framework should grant access to fundamental concepts about the biology underlying a given system, and it is these fundamental concepts that allow for a lucid dialogue among multiple fields. Here we present a framework that describes the relationship between DNA and DNA-binding proteins by considering the general factors that affect their interaction. Others have previously triangulated the relationships between DNA structure, DNA sequence, and protein–DNA interactions in various combinations, and the concepts presented here are informed by these earlier ideas.3a−3e