Sanford H. Leuba

Single molecule force spectroscopy in biology using the atomic force microscope.

The importance of forces in biology has been recognized for quite a while but only in the past decade have we acquired instrumentation and methodology to directly measure interactive forces at the level of single biological macromolecules and/or their complexes. This review focuses on force measurements performed with the atomic force microscope. A general introduction to the principle of action is followed by review of the types of interactions being studied, describing the main results and discussing the biological implications.

Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers

Single chromatin fibers were assembled directly in the flow cell of an optical tweezers setup. A single λ phage DNA molecule, suspended between two polystyrene beads, was exposed to a Xenopus laevis egg extract, leading to chromatin assembly with concomitant apparent shortening of the DNA molecule. Assembly was force-dependent and could not take place at forces exceeding 10 pN. The assembled single chromatin fiber was subjected to stretching by controlled movement of one of the beads with the force generated in the molecule continuously monitored with the second bead trapped in the optical trap. The force displayed discrete, sudden drops upon fiber stretching, reflecting discrete opening events in fiber structure. These opening events were quantized at increments in fiber length of ∼65 nm and are attributed to unwrapping of the DNA from around individual histone octamers. Repeated stretching and relaxing of the fiber in the absence of egg extract showed that the loss of histone octamers was irreversible. The forces measured for individual nucleosome disruptions are in the range of 20–40 pN, comparable to forces reported for RNA- and DNA-polymerases.

Proteins that specifically recognize cisplatin-damaged DNA: a clue to anticancer activity of cisplatin

Cisplatin, but not its trans geometric isomer, is a potent anticancer drug whose biological activity is a consequence of the formation of covalent adducts between the platinum compound and certain bases in DNA. Two classes of proteins have recently been identified that bind preferentially to damaged sites: proteins that specifically recognize those sites as a first step in their repair, and those that bind to such sites by virtue of structural similarity between the modified DNA and their own natural binding sites. Both classes of proteins may be involved, perhaps in opposing ways, in the cytotoxic effect of the drug.

Chromatin fiber structure: morphology, molecular determinants, structural transitions.

Despite more than 20 years of research, the structure of the chromatin fiber and its molecular determinants remain enigmatic. Recent developments in high-resolution microscopic techniques, as well as the application of mathematical modeling to chromatin fiber structure, have allowed the acquisition of some new insights into the structure and its determinants. Here we present some of the newest data on the structure of the chromatin fiber in both its extended and compacted states, and bring together this new knowledge with older data in an attempt to provide a unified view of how chromatin components interact with each other to form its various conformations. The structural transitions that are believed to take place during transcriptional activation and its cessation are also discussed. It becomes obvious that despite some progress in our understanding of the fiber structure and its dynamics, huge gaps continue to exist. Bridging these gaps will require further improvements in already available techniques and the introduction of completely new approaches.

Selective Requirements for Histone H3 and H4 N Termini in p300-Dependent Transcriptional Activation from Chromatin

The N-terminal tails of the core histones play important roles in transcriptional regulation, but their mechanism(s) of action are poorly understood. Here, pure chromatin templates assembled with varied combinations of recombinant wild-type and mutant core histones have been employed to ascertain the role of individual histone tails, both in overall acetylation patterns and in transcription. In vitro assays show an indispensable role for H3 and H4 tails, especially major lysine substrates, in p300-dependent transcriptional activation, as well as activator-targeted acetylation of promoter-proximal histone tails by p300. These results indicate, first, that constraints to transcription are imposed by nucleosomal histone components other than histone N-terminal tails and, second, that the histone N-terminal tails have selective roles, which can be modulated by targeted acetylation, in transcriptional activation by p300.

Linker Histone Tails and N-Tails of Histone H3 Are Redundant: Scanning Force Microscopy Studies of Reconstituted Fibers

The mechanisms responsible for organizing linear arrays of nucleosomes into the three-dimensional structure of chromatin are still largely unknown. In a companion paper (Leuba, S. H., et al. 1998. Biophys. J. 74:2823-2829), we study the contributions of linker histone domains and the N-terminal tail of core histone H3 to extended chromatin fiber structure by scanning force microscopy imaging of mildly trypsinized fibers. Here we complement and extend these studies by scanning force microscopy imaging of selectively reconstituted chromatin fibers, which differ in subtle but distinctive ways in their histone composition. We demonstrate an absolute requirement for the globular domain of the linker histones and a structural redundancy of the tails of linker histones and of histone H3 in determining conformational stability.

Assembly of single chromatin fibers depends on the tension in the DNA molecule: Magnetic tweezers study

We have used magnetic tweezers to study in real time chaperone-mediated chromatin assembly/disassembly at the level of single chromatin fibers. We find a strong dependence of the rate of assembly on the exerted force, with strong inhibition of assembly at forces exceeding 10 pN. During assembly, and especially at higher forces, occasional abrupt increases in the length of the fiber were observed, giving a clear indication of reversibility of the assembly process. This result is a clear demonstration of the dynamic equilibrium between nucleosome assembly and disassembly at the single chromatin fiber level.

DNA methylation-dependent chromatin fiber compaction in vivo and in vitro: requirement for linker histone

Dynamic alterations in chromatin structure mediated by postsynthetic histone modifications and DNA methylation constitute a major regulatory mechanism in DNA functioning. DNA methylation has been implicated in transcriptional silencing, in part by inducing chromatin condensation. To understand the methylation‐dependent chromatin structure, we performed atomic force microscope (AFM) studies of fibers isolated from cultured cells containing normal or elevated levels of m5C. Chromatin fibers were reconstituted on control or methylated DNA templates in the presence or absence of linker histone. Visual inspection of AFM images, combined with quantitative analysis of fiber structural parameters, suggested that DNA meth‐ylation induced fiber compaction only in the presence of linker histones. This conclusion was further substantiated by biochemical results.—Karymov, M. A., Tomschik, M., Leuba, S. H., Caiafa, P., Zlatanova, J. DNA methylation‐dependent chromatin fiber compaction in vivo and in vitro: requirement for linker histone. FASEB J. 15, 2631–2641 (2001)

Chromatin Fibers, One-at-a-time

Eukaryotic DNA is presented to the enzymatic machineries that use DNA as a template in the form of chromatin fibers. At the first level of organization, DNA is wrapped around histone octamers to form nucleosomal particles that are connected with stretches of linker DNA; this beads-on-a-string structure folds further to reach a very compact state in the nucleus. Chromatin structure is in constant flux, changing dynamically to accommodate the needs of the cell to replicate, transcribe, and repair the DNA, and to regulate all these processes in time and space. The more conventional biochemical and biophysical techniques used to study chromatin structure and dynamics have been recently complemented by an array of single-molecule approaches, in which chromatin fibers are investigated one-at-a-time. Here we describe single-molecule efforts to see nucleosomes, touch them, put them together, and then take them apart, one-at-a-time. The beginning is exciting and promising, but much more effort will be needed to take advantage of the huge potential that the new physics-based techniques offer.

Steric exclusion and wrapping of the excluded DNA strand occurs along discrete external binding paths during MCM helicase unwinding

The minichromosome maintenance (MCM) helicase complex is essential for the initiation and elongation of DNA replication in both the eukaryotic and archaeal domains. The archaeal homohexameric MCM helicase from Sulfolobus solfataricus serves as a model for understanding mechanisms of DNA unwinding. In this report, the displaced 5′-tail is shown to provide stability to the MCM complex on DNA and contribute to unwinding. Mutations in a positively charged patch on the exterior surface of the MCM hexamer destabilize this interaction, alter the path of the displaced 5′-tail DNA and reduce unwinding. DNA footprinting and single-molecule fluorescence experiments support a previously unrecognized wrapping of the 5′-tail. This mode of hexameric helicase DNA unwinding is termed the steric exclusion and wrapping (SEW) model, where the 3′-tail is encircled by the helicase while the displaced 5′-tail wraps around defined paths on the exterior of the helicase. The novel wrapping mechanism stabilizes the MCM complex in a positive unwinding mode, protects the displaced single-stranded DNA tail and prevents reannealing.