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Biochemistry Presentations

Presentations are listed in alphabetical order by the presenter's last name.

Determining the structural basis for recruitment of ALKBH3-ASCC repair complex on the proximal ubiquitin
Recognition and repair of DNA damage is required for proper cellular function. Recent studies show that the protein ASCC2, a subunit of the ALKBH3-ASCC DNA repair complex, locates sites of DNA damage by binding polyubiquitin chains assembled in proximity to the damage sites. It has been found that ASCC2 has an enhanced binding affinity to polyubiquitin chains despite only having one CUE domain, which normally binds to monoubiquitin. The cause of this enhanced binding affinity is unknown, leading to the hypothesis that a second, previously undiscovered ubiquitin binding site on ASCC2 exists. Binding models for the ASCC2:polyubiquitin complex place ubiquitin residues E64 and T66 at the novel binding interface. This binding model was tested by mutating ubiquitin residues E64 and T66 and measuring the binding affinity of the polyubiquitin mutant at the Wolberger lab at the Johns Hopkins University School of Medicine using isothermal titration calorimetry (ITC). It was found that binding affinity for this mutant polyubiquitin chain was decreased fourfold compared to the unmutated polyubiquitin chain. This leads to the conclusion that ubiquitin residues E64 and T66 are important for recognition by ASCC2. To determine if both E64 and T66 recognized, a ubiquitin mutant with just the E64 mutation was created. Utilizing funding obtained from the Summer Research Internship Award at Mount St. Mary’s University, residue E64 residue was mutated to alanine. The mutant was produced in E. coli and purified via acid precipitation. The E64 mutant will be incorporated into longer polyubiquitin chains and used in future ITC studies.
Presenter: Lauren Gray / Mentor: Patrick Lombardi, Ph.D.
Testing an ASCC2:diubiquitin binding model using isothermal titration calorimetry
Faithful detection and correction of DNA damage is necessary to prevent widespread genetic mutation and dysregulation of cellular processes. Recently, it has been shown that chains of the protein ubiquitin are assembled in proximity to sites of DNA alkylation damage to recruit the ALKBH3-ASCC DNA repair complex.  The main goal of this research project is to understand how the subunit ASCC2 of the ALKBH3-ASCC DNA repair complex recognizes polyubiquitin chains to initiate DNA repair. ASCC2 functions as the recruiting subunit that directly binds to polyubiquitin chains and recruits the other members of the DNA repair complex to begin DNA repair. The ASCC2 subunit recognizes polyubiquitin chains by binding to two linked ubiquitin proteins, binding sites are present on both the proximal and distal proteins of the complex, but are located on different amino acids within each protein. We simulated the binding affinity of ASCC2 to polyubiquitin chains using a diubiquitin complex. Research groups from Hopkins and other institutions have studied the binding affinities of ASCC2 to proposed binding sites on the ubiquitin proteins of the complex, however, while the interaction between ASCC2 and the distal ubiquitin of the complex is well characterized, how ASCC2 binds the proximal ubiquitin is unknown. This model was tested by mutating specific ASSC2 amino acids at the putative binding interface and comparing the binding affinity of the mutant and wild-type ASCC2 constructs to diubiquitin. Site-directed mutagenesis was used to change the amino acids at the binding interface to residues that would disrupt the interactions in the proposed binding model. In this experiment threonine 66 was mutated to alanine (T66A) due to its proximity to the binding interface. Perchloric acid precipitation and centrifugation were used to purify the mutant proximal ubiquitin for testing. Isothermal titration calorimetry was used to compare the binding affinity of wild type and mutant Y496D ASCC2 to K63-linked diubiquitin. This experiment showed that both constructs bind diubiquitin with approximately the same affinity. This indicated that amino acid Y496 is not a major contributor to the binding interface. Currently, the lab is working to synthesize enzymes that will be able to link single ubiquitins into diubiquitin chain on campus, so mutated complexes, such as T66A ASCC2, can be tested without having to travel to Baltimore to utilize technology at Hopkins.
Presenter: Abigail Hacker / Mentor: Patrick Lombardi, Ph.D.

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