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:
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, submitted). 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. Specific questions we are pursuing include:
- What is the composition and activity of the translesion replisome?
- How is the exchange of DNA polymerases at the replication fork mediated and regulated?
- How does the correct translesion polymerase synthesize over a specific lesion?
3) 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 aim to develop a multiplexed, microfluidic-based single-molecule platform to study DNA repair in cell-free extracts. These methods will be used to further explore the mechanistic details of the repair machinery and as a therapeutic and screening tool to help clinicians select effective chemotherapeutic treatments and assist in the search for novel inhibitors of DNA repair pathways.
- Observing polymerase exchange by simultaneous measurements of replisome composition and function at the single-molecule level. Loparo, J.J.; Kukczyk, A.W.; Richardson, C.C.; van Oijen, A.M.; (submitted) (2010).
- Single-molecule studies of the replisome. van Oijen, A.M. and Loparo, J.J. Ann. Rev. Biophys 39 (2010) 429-48.
- The PCNA sliding clamp uses two distinct sliding modes to move along DNA. Kochaniak, A.B.; Habuchi, S.; Loparo, J.J.; Chang, D.J.; Cimprich, K.A.; Walter, J.C.; van Oijen, A.M.; J. Biol. Chem. 284 (2009) 17700-10.
- Real-time single-molecule observation of rolling-circle DNA replication. Tanner, N.A*.; Loparo, J.J.*; Hamdan, S.M.; Jergic, S.; Dixon, N.E.; van Oijen, A.M.; Nucleic Acids Res. 37 (2009) e27. *equal contribution
- Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis. Hamdan, S.M.; Loparo, J.J.; Takahashi, M.; Richardson, C.C.; van Oijen, A.M.; Nature 457 (2009) 336-9.