Picture of Joe Loparo
Joseph John Loparo, Ph.D.
Associate Professor of Biological Chemistry and Molecular Pharmacology

The Loparo laboratory utilizes single-molecule methods to understand the dynamics of the multiprotein complexes involved in genome maintenance. We aim to develop approaches that allow us to probe the structure and function of these complexes in real time in order to better understand how their assembly and activity are regulated.

 

Research:

Single-Molecule Studies of DNA Damage Tolerance and Repair

The Loparo laboratory utilizes single-molecule methods to understand the dynamics of the multiprotein complexes involved in genome maintenance. We aim to develop approaches that allow us to probe the structure and function of these complexes in real time in order to better understand how their assembly and activity are regulated. Major areas of current research include:

Regulation of error prone DNA polymerases in DNA replication and translesion synthesis

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, allowing for rapid lesion bypass. In current work we are extending these studies to the fully reconstituted replisome and to studies in live cells.

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 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. We are currently working to further describe 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.

Molecular mechanisms of bacterial chromosome compaction and segregation

Bacteria use a collection of nucleoid-associated proteins to help condense and segregate their chromosomes. To better understand how these proteins remodel DNA, my laboratory has developed novel in vitro single-molecule approaches to better track DNA dynamics and visualize protein-DNA association in the absence of protein labeling. We have used these techniques to interrogate ParB and structural maintenance of chromosomes (SMC), two highly conserved proteins involved in chromosome organization and segregation. ParB is a sequence specific DNA binding protein that recognizes a 16 base pair sequence known as parS found near replication origins and spreads thousands of bases from these sites into neighboring non-specific DNA. In collaboration with David Rudner’s laboratory, we demonstrated that ParB spreading requires DNA bridging interactions both in vitro and in vivo and identified a patch of highly conserved arginine residues that are required for forming these higher order protein-DNA interactions.

SMCs are characterized by their unique V-shaped structure; SMC monomers dimerize at the apex of the V with each arm terminating in an ATPase head. Binding of ATP results in head-head engagement, which is believed to close the SMC ring. Central to understanding SMC function is elucidating the mechanism by which SMC interacts with and remodels DNA and determining how this activity is coupled to the SMC ATPase cycle. We have combined single-molecule imaging of fluorescently labeled prokaryotic SMC molecules with flow-stretching of individual DNAs to investigate how SMCs are loaded onto DNA and form higher order structures that condense DNA. Our work demonstrates that SMCs have varied interactions with DNA that evolve as SMCs are loaded and assembled on DNA. We are currently working to better understand how SMC clusters assemble both in vitro and in cells.

Address: 

Room SGM - 204A

250 Longwood Avenue

Boston, MA 02115

Publications View
The Gene-Silencing Protein MORC-1 Topologically Entraps DNA and Forms Multimeric Assemblies to Cause DNA Compaction.
Authors: Authors: Kim H, Yen L, Wongpalee SP, Kirshner JA, Mehta N, Xue Y, Johnston JB, Burlingame AL, Kim JK, Loparo JJ, Jacobsen SE.
Mol Cell
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The Role of Noncognate Sites in the 1D Search Mechanism of EcoRI.
Authors: Authors: Piatt SC, Loparo JJ, Price AC.
Biophys J
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A Flow-Extension Tethered Particle Motion Assay for Single-Molecule Proteolysis.
Authors: Authors: Drabek AA, Loparo JJ, Blacklow SC.
Biochemistry
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Protein translocation by the SecA ATPase occurs by a power-stroke mechanism.
Authors: Authors: Catipovic MA, Bauer BW, Loparo JJ, Rapoport TA.
EMBO J
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Guidelines for DNA recombination and repair studies: Mechanistic assays of DNA repair processes.
Authors: Authors: Klein HL, Ang KKH, Arkin MR, Beckwitt EC, Chang YH, Fan J, Kwon Y, Morten MJ, Mukherjee S, Pambos OJ, El Sayyed H, Thrall ES, Vieira-da-Rocha JP, Wang Q, Wang S, Yeh HY, Biteen JS, Chi P, Heyer WD, Kapanidis AN, Loparo JJ, Strick TR, Sung P, Van Houten B, Niu H, Rothenberg E.
Microb Cell
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A single XLF dimer bridges DNA ends during nonhomologous end joining.
Authors: Authors: Graham TGW, Carney SM, Walter JC, Loparo JJ.
Nat Struct Mol Biol
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Observing Bacterial Chromatin Protein-DNA Interactions by Combining DNA Flow-Stretching with Single-Molecule Imaging.
Authors: Authors: Kim H, Loparo JJ.
Methods Mol Biol
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Single-molecule imaging reveals multiple pathways for the recruitment of translesion polymerases after DNA damage.
Authors: Authors: Thrall ES, Kath JE, Chang S, Loparo JJ.
Nat Commun
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A network of cis and trans interactions is required for ParB spreading.
Authors: Authors: Song D, Rodrigues K, Graham TGW, Loparo JJ.
Nucleic Acids Res
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Ensemble and Single-Molecule Analysis of Non-Homologous End Joining in Frog Egg Extracts.
Authors: Authors: Graham TGW, Walter JC, Loparo JJ.
Methods Enzymol
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