November 19th: Yuxi Li (Hengyao Niu) "Mechanism of DNA Structural Sensitivity Of Saccharomyces cerevisiae Exo1 Nuclease in DNA Damage Repair" 10:00am
Defense will be held virtually. To Register For The Defense Please Click On The Zoom Link
Exonuclease 1 (Exo1), is a eukaryote specific nuclease that is significant in maintaining genomic integrity and conserved from yeast to humans. Exo1 utilizes its 5’ to 3’ double-stranded DNA exonuclease activity to remove nucleotides from the 5' end of DNA duplex, which functions in resecting double-strand breaks and removing mismatches in cells. Although the activity of Exo1 on canonical dsDNA has been well-studied, less is known about the detailed mechanism of Exo1 processing structured DNA during DNA metabolism. First, we determined that Exo1 requires a minimum of four-base-pair duplex region to exert nuclease activity. A conserved residue, Lysine 185, on Exo1 that contacting the phosphate group bridging the third and fourth nucleotide on the digestion strand is responsible to control the nuclease activity of Exo1 by influencing the DNA duplex binding capability of Exo1. Hence, DNA duplex binding is essential for Exo1 when the 3’ overhanging ssDNA is short or when single-stranded binding protein RPA occupies the 3’ overhanging end. Moreover, we found Exo1 nuclease has sensitivity towards structured DNA. Although Exo1 is the sole nuclease found functioning in mismatch repair, we found Exo1 tolerates base substitution, single nucleotide insertion or deletion but is sensitive to DNA loops or hairpin structures that consist of dinucleotide microsatellite, trinucleotide repeat and inverted repeat sequences in vitro. In humans, the expansion of trinucleotide repeats sequence causes neurological diseases, such as Huntington’s disease. Using trinucleotide repeats as a guidance, we found Dna2 nuclease processes the hairpin together with Exo1 to alleviate the pausing caused by DNA structures in the presence of single-stranded binding protein RPA. We also reported that Dna2 physically interacts with Exo1. Therefore, we proposed a model that Exo1 and Dna2 nucleases that are previously believed to function in parallel during DNA end resection synergize to process structured DNA during DNA metabolism.
Dec 12: Garrett Booher's (VanNieuwenhze lab) "Fluorescent D-amino acids as tools for studying peptidoglycan synthase activity and developing a high-throughput screening assay to identify novel antibiotics" at 4:00 pm
Peptidoglycan is a unique structure to bacteria, which allows this structure to be an important target for antibiotics due to its orthogonality to human cell biology. One target of such antibiotics is the bacterial transpeptidases, mainly penicillin binding proteins (PBPs), that have been widely studied since the discovery of penicillin in the early 1900's. However, tools to study the dynamics of these enzymes in vivo and in vitro have been lacking. Recently, the VanNieuwenhze lab discovered that transpeptidases can utilize fluorescent D-amino acids (FDAAs) to incorporate a fluorescent tag into peptidoglycan. Thus, resulting in the labeling of peptidoglycan to study the growth modes of various different bacterial species. These FDAAs have been applied to a wide range of studies, including dynamics of peptidoglycan synthesis, peptidoglycan-enzyme interactions, and peptidoglycan metabolism. Herein, I report additional applications of FDAAs to further the understanding of the enzymes involved in peptidoglycan processes. Specifically, the development of several methods for studying the enzymatic reactions of various transpeptidases. Furthermore, an assay developed to study PBPs in vitro in real-time is reported. This assay has been optimized to the point that a robust screening of natural products was performed, and several hits were identified. Though the positive hits did not meet the threshold for clinical drugs, chemical modification of the compounds could enhance activity. Specifically, these chemical compounds can be modified to incorporate a D-amino acid backbone. The hypothesis being that since the enzymes involved in peptidoglycan synthesis can use FDAAs, substituting the fluorophore with a reactive compound could lead to a novel class of antibiotics. This work demonstrates the value of such an assay to screen for compounds. These can be further optimized to allow new and potent antibiotics to be discovered and implemented as therapeutics.