Poster abstracts

Poster number 10 submitted by Caroline Karbowski

Understanding a DNA Repair Protein (LiRecT) With a Mutational Analysis

Caroline F. Karbowski (Department of Biological Chemistry and Pharmacology, The Ohio State University), Charles E. Bell (Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology, The Ohio State University), Brian J. Caldwell (Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology, The Ohio State University), Alyssa M. Wiegand (Department of Biological Chemistry and Pharmacology, The Ohio State University)

Abstract:
Some bacteriophage encode a recombinase that catalyzes single-stranded DNA annealing (SSA), a DNA repair process (Kuzminov, 1999). These proteins are distantly related to RAD52, the primary human SSA protein and a target for cancer therapeutics (Reddy et al., 1997). While there are structures of RAD52 with single-stranded DNA (ssDNA), there is no structure of RAD52 with two complementary strands of ssDNA bound simultaneously, and its mechanism of action is still unknown (Zakharova et al., 2021). We used a single particle cryo-electron microscopy (cryo-EM) to determine a 3.4 Å structure of a RAD52 homolog, LiRecT, from a prophage of Listeria innocua in complex with two complementary 83mer oligonucleotides. The structure reveals a helical protein filament with a narrow outer groove for binding a DNA duplex that is highly extended and unwound. To test the functional importance of the residues contacting the DNA in the structure, 42 mutations on 21 residues were mutated to alanine and/or other residue types. The mutants were purified using small-scale nickel spin column purification and their ability to bind to DNA was tested with a gel shift assay where free and bound DNA were separated and visualized. Some of the mutations to residues contacting the inner DNA strand, or residues at the subunit interface drastically decreased binding, while mutations to residues contacting the outer strand and the wedge did not (Caldwell et al., 2022). A more quantitative ssDNA binding assay (fluorescence polarization) is currently being conducted for each mutant. These data provide insights into the mode of DNA binding and the mechanism of protein-catalyzed SSA.

References:
Caldwell, B. J., et al. (2022). Structure of a RecT/Redβ family recombinase in complex with a duplex intermediate of DNA annealing. Nature Communications, 13(1). https://doi.org/10.1038/s41467-022-35572-z
Kuzminov, A. (1999). Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda. Microbiol. Mol. Biol. Rev. 63, 751–813.
Reddy, G., et al. (1997). Human RAD52 protein promotes single-strand DNA annealing followed by branch migration. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 377(1), 53-59. doi:10.1016/s0027-5107(97)00057-2
Zakharova, K., et al. (2021). Mutational analysis of Redβ single strand annealing protein: Roles of the 14 lysine residues in DNA binding and recombination in vivo. International Journal of Molecular Sciences, 22(14), 7758. doi:10.3390/ijms22147758

Keywords: Cryoelectron microscopy, DNA-binding proteins, DNA Repair