Studying multi-protein complexes at the single-molecule level provides additional challenges as compared to single enzymes. The Kd’s that govern the association of the various protein components are often much higher than the concentrations permissible for single-molecule imaging. We aim to develop generalized approaches to studying fluorescently labeled proteins at physiological concentrations through the application of photoswitchable fluorophores and nanophotonics. Additionally, single-molecule assays capable of correlating structure and function are critical in describing the dynamics of multi-protein machines.
Single-Molecule Studies of DNA Damage Tolerance and Repair
Our laboratory is interested in developing and applying single-molecule methods to better understand the molecular dynamics of multi-protein complexes that carry out duplication, maintenance and transmission of the genome. Traditional ensemble or bulk biochemistry has provided remarkable insight into the various activities of individual proteins and their collective action in these complexes. However, probing the dynamics of protein-protein interactions is extremely difficult in bulk experiments as the stochastic appearance and disappearance of transient intermediates tends to obscure any observable when averaged over the ensemble. Single-molecule methods are a powerful new way to overcome this problem by observing the individual trajectories of proteins as they function. Major areas of current research include:
Bacteria typically store their genetic information in a single circular chromosome that is several million DNA bases long. In order to maintain and duplicate this chromosome, called the nucleoid, bacteria must accomplish two major feats of structural engineering: First, a giant 1.5 millimeter-long DNA molecule must be packaged into a bacterial cell that is over a thousand times shorter. Second, newly replicated sister chromosomes must be disentangled and separated without the advantage of the sophisticated mitotic machinery that is present in eukaryotic cells. Work over the last several decades has identified a number of nucleoid-associated proteins (NAPs) that play essential roles in these processes, yet it remains unclear how various NAP-DNA interactions collectively regulate nucl
We are interested in how cells regulate the access of low-fidelity polymerases to the replication fork as their misuse leads to genome instability. In translesion synthesis (TLS), error-prone TLS polymerases are recruited to sites of DNA damage to carry out strand extension over DNA lesions that block the progress of the replisome. Using the E. coli replisome as a model system, we have demonstrated that we can reconstitute translesion synthesis at site-specific DNA lesions and observe polymerase exchange on individual DNAs. Using this approach we have shown that the translesion polymerases Pol IV and Pol II can bind the processivity clamp beta, allowing for rapid lesion bypass.
DNA double strand breaks (DSBs) are extremely toxic lesions that can arise spontaneously or can be induced by agents such as ionizing radiation or endonucleases involved in programmed genome rearrangements. For the majority of the cell cycle DSBs are repaired by NHEJ, a process that robustly ligates even damaged or incompatible DNA ends, albeit in a way that often generates insertion or deletion mutations. We are using single-molecule FRET approaches to directly visualize the repair of DSBs in reconstituted systems and in vertebrate cell free extracts. We have demonstrated that end synapsis passes through at least two structurally distinct states and have identified the core NHEJ factors required to form these states.