Allosteric effects of E. coli SSB and RecR proteins on RecO protein binding to DNA

Escherichia coli single stranded (ss) DNA binding protein (SSB) plays essential roles in DNA maintenance. It binds ssDNA with high affinity through its N-terminal DNA binding core and recruits at least 17 different SSB interacting proteins (SIPs) that are involved in DNA replication, recombination, and repair via its nine amino acid acidic tip (SSB-Ct). E. coli RecO, a SIP, is an essential recombination mediator protein in the RecF pathway of DNA repair that binds ssDNA and forms a complex with E. coli RecR protein. Here, we report ssDNA binding studies of RecO and the effects of a 15 amino acid peptide containing the SSB-Ct monitored by light scattering, confocal microscope imaging, and analytical ultracentrifugation (AUC). We find that one RecO monomer can bind the oligodeoxythymidylate, (dT)15, while two RecO monomers can bind (dT)35 in the presence of the SSB-Ct peptide. When RecO is in molar excess over ssDNA, large RecO-ssDNA aggregates occur that form with higher propensity on ssDNA of increasing length. Binding of RecO to the SSB-Ct peptide inhibits RecO-ssDNA aggregation. RecOR complexes can bind ssDNA via RecO, but aggregation is suppressed even in the absence of the SSB-Ct peptide, demonstrating an allosteric effect of RecR on RecO binding to ssDNA. Under conditions where RecO binds ssDNA but does not form aggregates, SSB-Ct binding enhances the affinity of RecO for ssDNA. For RecOR complexes bound to ssDNA, we also observe a shift in RecOR complex equilibrium towards a RecR4O complex upon binding SSB-Ct. These results suggest a mechanism by which SSB recruits RecOR to facilitate loading of RecA onto ssDNA gaps

Are the intrinsically disordered linkers involved in SSB binding to accessory proteins

Tuesday, February 18, 2020, 12:30 p.m., 1130 Eck Hall of Law Speakers: Josh Divine and Marah Stith McLeod Federal law regularly incorporates state law as its own, but meaningful use of such dynamic incorporation in criminal law is very rare. Its use in the criminal law context could reduce the political inertia that makes reforming criminal laws exceptionally difficult. It could also serve federalism by giving state legislatures the opportunity to exercise greater oversight of enforcement discretion, thereby enhancing enforcement accountability.https://scholarship.law.nd.edu/ndls_posters/1472/thumbnail.jp

A disordered region controls cBAF activity via condensation and partner recruitment

Intrinsically disordered regions (IDRs) represent a large percentage of overall nuclear protein content. The prevailing dogma is that IDRs engage in non-specific interactions because they are poorly constrained by evolutionary selection. Here, we demonstrate that condensate formation and heterotypic interactions are distinct and separable features of an IDR within the ARID1A/B subunits of the mSWI/SNF chromatin remodeler, cBAF, and establish distinct sequence grammars underlying each contribution. Condensation is driven by uniformly distributed tyrosine residues, and partner interactions are mediated by non-random blocks rich in alanine, glycine, and glutamine residues. These features concentrate a specific cBAF protein-protein interaction network and are essential for chromatin localization and activity. Importantly, human disease-associated perturbations in ARID1B IDR sequence grammars disrupt cBAF function in cells. Together, these data identify IDR contributions to chromatin remodeling and explain how phase separation provides a mechanism through which both genomic localization and functional partner recruitment are achieved

Specificity of SSB binding to its Interacting Proteins and Multiple Allosteric Effects of SSB C-terminal Tail on Assembly and DNA Binding of E. coli RecOR Proteins

The homo-tetrameric E. coli single strand (ss) DNA binding (SSB) protein is an essential component in DNA maintenance for its role in binding and protecting single stranded DNA intermediates via its N-terminal DNA binding domain (DBD). SSB also acts as a hub to recruit at least 17 SSB interacting proteins (SIPs) involved in DNA replication, recombination, and repair via its 9 amino acid C-terminal acidic tip region. A 56 amino acid intrinsically disordered linker connects the DBD and the acidic tip and plays a role in cooperative binding to ssDNA. Using isothermal titration calorimetry, I determined that the SSB-Ct peptides bind to different SIPs with specificity, with affinity decreasing in the order: RecO \u3e PriA ~ c subunit of DNA Pol III \u3e PriC. I also determined that the intrinsically disordered linker itself does not contribute to SIP binding. There are, however, additional interactions between the DBD of SSB and RecO. Furthermore, as the acidic tip region can interact intramolecularly with the DBD of SSB, RecO is in competition with the DBD to bind to the acidic tip. SSB-Ct binding to DBD is eliminated when the DBD is occupied by ssDNA. E. coli RecO is a recombination mediator protein (RMP) that is essential in one of the two major pathways of DNA repair, and interacts with an accessory protein, RecR. I investigated the oligomeric assembly properties of E. coli RecO and RecR proteins as well as the RecOR complexes using analytical ultracentrifugation, both by sedimentation velocity and sedimentation equilibrium. I found that E. coli RecR exists in a pH-dependent dimer-tetramer equilibrium, and that the dimeric state is stabilized at higher pH. However, I found that monomeric RecO forms complexes with only the RecR tetramer forming two RecOR complex species, RecR4O and RecR4O2. Investigating the DNA binding activity of RecO, I observed with light scattering measurements and confocal microscopy imaging that RecO-DNA complexes form aggregates with the propensity to form aggregates increasing with the length of DNA. Binding of RecO to either an SSB-Ct containing peptide or RecR inhibits aggregate formation. The direct interactions between SSB and SIPs have been shown to influence various activities of the SIPs. I observed multiple allosteric effects of SSB-Ct peptides on RecOR complex formation and RecO(R) binding to ssDNA. While E. coli RecR does not interact with SSB or DNA, I found that SSB-Ct peptides stabilize the RecR4O complex over R4O2. Furthermore, SSB-Ct peptides allosterically enhance binding of RecO to ssDNA but reduce binding of RecOR complex to ssDNA. From these observations, I suggest a mechanism by which SSB recruits RecO and RecR proteins to a damaged DNA site facilitating the loading of RecA protein to initiate homologous recombination

Fabrication and Characterization of Graphene-Boron Nitride van der Waals Heterostructure

Since the quantum Hall effect of hybrid graphene is impeded by its usual silicon dioxide substrate, the objective of this project is to stack hybrid graphene onto isoelectronic few-atom thick hexagonal boron nitride (hBN) lattices, constructing a van der Waals heterostructure. The electronic properties measurement of the heterostructure at low temperature and high magnetic field up to 9T yielded improved quality of quantum Hall resistance data. However, the visibility of graphene is compromised with the new BN substrate, the second part of this project is to investigate spectroscopic methods to inverse image graphite using a home-built Raman microscope

Allosteric effects of SSB C-terminal tail on assembly of E. coli RecOR proteins

Escherichia coli RecO is a recombination mediator protein that functions in the RecF pathway of homologous recombination, in concert with RecR, and interacts with E. coli single stranded (ss) DNA binding (SSB) protein via the last 9 amino acids of the C-terminal tails (SSB-Ct). Structures of the E. coli RecR and RecOR complexes are unavailable; however, crystal structures from other organisms show differences in RecR oligomeric state and RecO stoichiometry. We report analytical ultracentrifugation studies of E. coli RecR assembly and its interaction with RecO for a range of solution conditions using both sedimentation velocity and equilibrium approaches. We find that RecR exists in a pH-dependent dimer-tetramer equilibrium that explains the different assembly states reported in previous studies. RecO binds with positive cooperativity to a RecR tetramer, forming both RecR4O and RecR4O2 complexes. We find no evidence of a stable RecO complex with RecR dimers. However, binding of RecO to SSB-Ct peptides elicits an allosteric effect, eliminating the positive cooperativity and shifting the equilibrium to favor a RecR4O complex. These studies suggest a mechanism for how SSB binding to RecO influences the distribution of RecOR complexes to facilitate loading of RecA onto SSB coated ssDNA to initiate homologous recombination