Abstract:

Over the past few decades, there has been steady progress in both our ability to produce biological material and in our ability to manipute matter at small length scales. These two developments merge in a fascinating area of confluence called single-molecule biophysics in which an understanding of biological matter from physical principles becomes possible. I will illustrate the development of this interdisciplinary field and show how several newly-developed techniques allow us to shed light on genomic processes such as DNA compaction, replication, and transcription.

 

In our studies of DNA compaction, we have monitored the real-time loading of tetramers or complete histone octamers onto DNA by Nucleosome Assembly Protein-1 (NAP1) by measuring the twist and length of single DNA molecules. Remarkably, we find that tetrasomes exhibit spontaneous flipping between a preferentially occupied left-handed state and a right-handed state, separated by free energy difference of 2.3 k_BT (1.5 kcal/mol). The application of weak positive torque converts left-handed tetrasomes into right-handed tetrasomes, whereas nucleosomes display more gradual conformational changes. These findings reveal unexpected dynamical rearrangements of the nucleosomal structure, suggesting that chromatin can serve as a ‘‘twist reservoir,’’ offering a mechanistic explanation for the regulation of DNA supercoiling in chromatin.

In our studies of DNA replication, we have examined the termination of replication in Escherichia coli, where replisome progression beyond the termination site is prevented by Tus proteins bound to asymmetric Ter sites. Structural evidence indicates that strand separation on the blocking (non-permissive) side of Tus–Ter triggers roadblock formation, but biochemical evidence also suggests roles for protein- protein interactions. Our DNA unzipping experiments demonstrate that nonpermissively oriented Tus–Ter forms a tight lock in the absence of replicative proteins, whereas permissively oriented Tus–Ter allows nearly unhindered strand separation. Quantifying the lock strength reveals the existence of several intermediate lock states that are impacted by mutations in the lock domain but not by mutations in the DNA-binding domain. Lock formation is highly specific and exceeds reported in vivo efficiencies. We therefore postulate that protein-protein interactions may actually hinder, rather than promote, proper lock formation.

Our studies of DNA replication termination benefit from the use of high-throughput single-molecule techniques, and in concluding I will briefly illustrate how these approaches can also be used to investigate polymerase mechanisms.  Most recently, this has allowed us to study RNA-dependent RNA polymerases and in particular their error incorporation, a process whose frequency is important in viral defense and evolution.