Joseph J. Loparo, Ph.D.
Department of Biological Chemistry and Molecular Pharmacology
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:
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?
3) Dissecting DNA Repair Pathways in Eukaryotes
Hamdan, S.M.; Loparo, J.J.; Takahashi, M.; Richardson, C.C.; van Oijen, A.M. (2009) Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis. Nature 457: 336-9.
Tanner, N.A*.; Loparo, J.J.*; Hamdan, S.M.; Jergic, S.; Dixon, N.E.; van Oijen, A.M. (2009) Real-time single-molecule observation of rolling-circle DNA replication. Nucleic Acids Res. 37: e27. *equal contribution
van Oijen, A.M and Loparo, J.J. (2010) Single-molecule studies of the replisome. Ann. Rev. Biophy.s 39: 429-48.
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