The nucleus of a eukaryotic cell is a highly dynamic reaction center for many DNA-based cellular processes, as well as an effective compaction center for DNA storage. The basic packing unit of chromatin is the nucleosome, and even this lowest level of genomic compaction presents a strong barrier to many DNA-binding cellular factors. In a coordinated and regulated fashion, DNA associated with nucleosomes must be made transiently accessible to permit many essential processes, such as transcription, DNA replication, recombination, and repair. Many of the enzymes involved in these processes are known to be processive molecular motors that utilize chemical energy derived from nucleoside triphosphate (NTP) hydrolysis to translocate along and rotate around DNA. Fundamental questions remain regarding the physical properties and mechanisms of genetic information storage, retrieval, and duplication, and in particular the regulation of nucleosome packing/unpacking and the mechanisms of DNA-based molecular motors involved in various cellular processes.
We take systematic, multipronged, and interdisciplinary approaches to investigate these processes, which are kinetic and mechanical in nature, involving translocation, rotation, force, and torque. We combine single-molecule studies, biochemical and molecular biological studies, theoretical modeling, and novel instrument development to address these problems.
Development of Novel Biophysical Techniques
Our lab has made several technical innovations that expand the capabilities of single-molecule manipulation and detection. Two in particular are beginning to have a significant impact.
We have developed a DNA-unzipping technique as a versatile and powerful single-molecule method to probe protein-DNA interactions. Using an optical trap, we unzip a single DNA double helix in the presence of DNA-binding proteins. When the unzipping fork reaches a bound protein molecule, a dramatic increase in the unzipping force is detected, followed by a sudden force reduction as the interaction is disrupted. Analysis of the unzipping force of an unbinding event reveals the spatial location and the strength of the interaction. We have applied this approach to study restriction enzymes, DNA repair enzymes, and nucleosomes. Recently we demonstrated that we are able to detect the location of a bound protein with near single–base-pair resolution, accuracy, and precision.
Despite the importance of torque to biology, direct measurements of torque have proved to be challenging. We have developed an angular optical trapping instrument that permits simultaneous and direct measurements of force and torque for concurrent observation of the tensile and torsional properties of biological molecules over broad ranges of forces and torques. In an angular optical trap, four signals are simultaneously and directly measured: torque, rotation, force, and position, all with high spatial and temporal resolution. This wide bandwidth is also well suited for detection of highly kinetic processes. To ensure controlled orientation of the trapping particle and its specific attachment to the molecule of interest, we have nanofabricated quartz cylinders that have proved to be ideal trapping particles. The use of these cylinders has dramatically enhanced the precision of torque measurements, making the angular optical trap a powerful tool for biological torque measurements. This instrument is opening up new possibilities for experiments on biological molecules, many of which are known to generate rotational motions and work against topological obstacles (e.g., topoisomerases, RNA polymerases).
Mechanical Stability of Nucleosomes
We have conducted extensive studies on the mechanics of nucleosome unpacking and remodeling. We have opened many new avenues for nucleosome research by providing distinct mechanical signatures for nucleosome disruption, precise dynamic signatures of nucleosome structure, and single-molecule measurements of nucleosome remodeling.
By stretching single DNA molecules containing nucleosomes, we have located the strong protein-DNA interactions within a nucleosome and revealed that the mechanical disruption of the nucleosome occurs in three stages. We also demonstrated how different histone tails and their acetylation regulate the mechanical stability of nucleosomes. By unzipping DNA molecules containing a single nucleosome, we have located the absolute positions of strong interactions within a nucleosome to near base-pair accuracy. We have used this approach to determine the change in location and structure of a nucleosome after remodeling and after transcription. This DNA-unzipping technique provides a unique and novel single-molecule approach to study nucleosome structures at high resolution. We anticipate that it will be a powerful tool to study histone modifications, nucleosome dynamics, and nucleosome regulation of gene expression. By twisting DNA molecules containing a single nucleosome, we are investigating the regulation of nucleosome stability under torque.
Mechanism of Transcription
RNA polymerase is the key enzyme involved in transcription, an essential process in gene expression and its regulation. During transcription, RNA polymerase translocates processively along a DNA template while incorporating nucleotides into the nascent transcript RNA. We have shown that RNA polymerase is a powerful molecular motor by studying transcription in vitro, both mechanically and at the level of single molecules. We have formulated and tested the first sequence-dependent kinetic models for transcription elongation based on thermodynamics and statistical mechanics. We have established a thermal ratchet model that is able to predict locations of sequence-dependent transcription pauses and force-velocity relations (without any free parameters), lending strong support to a simple mechanochemical coupling mechanism. The modeling work serves as a foundation to understand how RNA polymerase transcribes through obstacles such as nucleosomes.
Unwinding of DNA by Helicase
During DNA replication, the two strands of double-stranded DNA (dsDNA) must first be separated into single-stranded DNA (ssDNA). Helicases are vital enzymes that carry out this function. One intriguing question is how helicase couples its translocation along ssDNA to unwinding of dsDNA. Does helicase passively wait for thermal opening of the DNA fork before its forward translocation, or will it actively destabilize the fork to facilitate fork opening? We have addressed this question by directly monitoring the motion of T7 helicase as it translocates along ssDNA and unwound dsDNA. We have provided evidence that T7 helicase possesses a DNA sequence- and force-dependent unwinding rate. This work, in conjunction with theoretical modeling, led us to conclude that T7 helicase uses an active mechanism to unwind DNA and provides a quantitative approach to determine if a helicase unwinds DNA passively or actively, and if active, the degree of activeness. Our work also suggests a mechanism by which helicase may work with accessory proteins for efficient DNA replication.
Direct Biological Torque Measurements
The bending and torsional properties of DNA influence numerous cellular processes, notably DNA compaction, transcription, replication, and protein-DNA binding. The most concise way to characterize the tensile and torsional response of DNA is the force-torque phase diagram. Using the angular optical trap, we have mapped out many regions of the phase diagram. We have observed the DNA phase transition from B to supercoiled P form, the DNA-buckling transition during supercoiling, and the DNA-melting transition.
