Joseph Loparo

Harvard Medical School
Dept of Biological Chemistry and Molecular Pharmacology
240 Longwood Ave SGM 204B
Boston, MA 02115
Tel: 617-432-5586
Email: joseph_loparo@hms.harvard.edu

Website:
https://loparo.hms.harvard.edu/
Lab Size: Between 5-10

Summary

The laboratory is primarily focused on developing and applying single-molecule methods to better understand the molecular dynamics of the multi-protein complexes that are involved in genome maintenance.   Areas of current interest include:

Regulation of translesion synthesis
DNA damage acts as a potent block to the replication machinery. Error-prone translesion synthesis (TLS) is one pathway utilized to overcome this challenge.  In TLS, translesion polymerases are recruited to sites of DNA damage to carry out strand extension over DNA lesions enabling resumption of DNA replication. Using the bacterial replisome as a model, we are studying how TLS polymerases are recruited to the replisome, how protein-protein interactions control their access to DNA and how other factors expressed during the SOS damage response might further regulate their activity.

Repair of double-strand DNA breaks by non-homologous end joining
DNA double strand breaks (DSBs) are extremely toxic lesions that can arise spontaneously or 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 aim to understand how the assembly of the NHEJ machinery is controlled, how DNA ends are held together by this machinery, and how mutations are minimized during repair.

The role of nucleoid associated proteins in bacterial chromosome organization
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.  We are developing single-molecule approaches to directly probe how nucleoid associated proteins condense DNA.

Publications

Multistep assembly of DNA condensation clusters by SMC. Kim, H.; Loparo, J.J.
Nat Commun (Accepted 2015)

Mechanical allostery: Evidence for a force requirement in the proteolytic activation of Notch. Gordon, W.R.; Zimmerman, B.; He, L.; Miles, L.J.; Huang, J.; Tiyanont, K.; McArthur, D.G.; Aster, J.C.; Perrimon, N.; Loparo, J.J.*, Blacklow, S.C.*  Dev. Cell 33 (2015) 729-736.

ParB spreading requires DNA bridging. Graham, T.G.W.; Wang, X.; Song, D.; Etson, C.M.; van Oijen, A.M.; Rudner, D.Z.; Loparo, J.J. Genes Dev 28 (2014) 1228-38.

Polymerase exchange on single DNA molecules reveals processivity clamp control of translesion synthesis. Kath, J.E,; Jergic, S.; Heltzel, J.M.H.; Jacob, D.T.; Dixon, N.E.; Sutton, M.D.; Walker, G.C.; Loparo, J.J. Proc. Natl. Acad. Sci. USA 111 (2014) 7647-52