Biological Chemistry and Molecular Pharmacology
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.
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.
Nature structural & molecular biology, September 3, 2018
Methods in molecular biology (Clifton, N.J.), January 1, 2018
Nature communications, December 18, 2017
Nucleic acids research, July 7, 2017
Methods in enzymology, May 15, 2017
Nucleic acids research, May 16, 2016
Molecular cell, March 17, 2016
Nature communications, March 8, 2016
Nature communications, January 4, 2016
Nucleic acids research, December 10, 2015