Single-Molecule Studies of Genome Maintenance

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:

1) Developing new single-molecule tools to study multi-protein complexes

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. In recent work, we have demonstrated a powerful new assay that combines nanomanipulation of DNA with observation of fluorescently labeled proteins to measure the activity and composition of the replisome, the multi-protein complex that carries out DNA replication (Loparo et al, PNAS 2011). This assay is broadly applicable to any number of multi-protein complexes acting on DNA and future efforts will build on this work by focusing on improving structural sensitivity and spatial resolution.

2) Structure, function and regulation of the translesion replisome

Cell survival requires both an ability to repair DNA damage and to tolerate it. In collaboration with Graham Walker's laboratory at MIT's Dept. of Biology, we are applying single-molecule methods to characterize the translesion polymerases of E. coli. Translesion polymerases are specialized DNA polymerases capable of synthesizing over certain DNA lesions that stall the replicative DNA polymerase. Given that these polymerases can be mutagenic, we are most interested in how specific protein-protein interactions between regulatory proteins and the replisome mediate the exchange of replicative and translesion polymerases.

3) Compaction and segregation of the bacterial chromosome

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 nucleoid architecture. In collaboration with David Rudner's lab here at HMS, we are investigating the molecular mechanisms by which nucleoid-associated proteins from the model gram-positive bacterium Bacillus subtilis organize and segregate the bacterial chromosome.

4) Dissecting DNA repair pathways in eukaryotes

Deficiencies in DNA repair lead to a number of serious diseases, including cancer. To address the complexity of DNA repair in humans, we are both reconstituting repair reactions in vitro and using cell free extracts. We aim to further the current mechanistic understanding of repair reactions by visualizing the composition of the repair machinery in real time and correlating this with its repair proficiency. Current areas of study include base excision repair and homologous recombination.