Genome-wide investigation of maize RAD51 binding affinity through phage display

Authors: MILSTED, CLAIRE - Arizona State University; DAI, BO - University Of Minnesota; GARCIA, NELSON - University Of Minnesota; YIN, LU - Arizona State University; HE, YAN - Cornell University; Kianian, Shahryar; PAWLOWSKI, WOJCIECH - Cornell University; CHEN, CHANGBIN - Arizona State University

Submitted to: BMC Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/18/2022
Publication Date: 3/12/2022
Citation: Milsted, C., Dai, B., Garcia, N., Yin, L., He, Y., Kianian, S., Pawlowski, W., Chen, C. 2022. Genome-wide investigation of maize RAD51 binding affinity through phage display. BMC Genomics. 23. Article 199. https://doi.org/10.1186/s12864-022-08419-6.
DOI: https://doi.org/10.1186/s12864-022-08419-6

Interpretive Summary: The DNA double-strand break repair protein RAD51 is conserved in all eukaryotes but was initially identified in Saccharomyces cerevisiae. It enables double-strand break repair by homologous recombination, specifically catalyzing the alignment of the broken DNA ends to a double-stranded template, a process known as strand invasion. In many eukaryotes including humans and Arabidopsis, RAD51-mediated DNA repair requires the DNA repair protein BRCA2. Mutations in the BRCA2 gene are risk factors for breast and ovarian cancer in humans. This work describes the use of phage display to identify proteins that interact with maize RAD51A1.These results reveal several peptides which bind to maize protein and support a potential role for maize RAD51A1 in transcriptional regulation and plant defense.

Technical Abstract: RAD51 proteins, which are conserved in all eukaryotes, repair DNA double-strand breaks. This in turn is critical to chromosome crossover in meiosis, chromosome pairing, and, in many species, successful reproduction. Many rad51 mutant plants are sterile or have low seed set. Work in Arabidopsis suggests that RAD51 plays a role in plant defense, including binding to the promoter of the defense gene PR1. However, this second function of RAD51 has not been investigated in maize. Phage display is a tool that allows discovery of novel protein-protein interactions. It is used in biomedical contexts including antibody development but has further potential as a tool for basic science, including in non-medical fields such as plant biology. Maize RAD51A1 was screened against a random phage library. Eleven peptide sequences were recovered from 15 phages which bound ZmRAD51A1 in vitro; three sequences were found in multiple successfully binding phages. Nine of these phage interactions were verified in vitro through ELISA and/or dot blotting. BLAST searches did not reveal any maize proteins which contained the exact sequence of any of the selected phage peptides, although one of the selected phages had a strong alignment (E-value = 0.01) to a possible binding domain of maize BRCA2. With the exception of phage peptide #11, believed to be a false positive, there were no perfect matches to proteins from other species. Therefore, we designed 32 additional short peptides using amino acid sequences found in the predicted maize proteome which closely but imperfectly align to the selected phage peptides. Of these synthesized peptides, 14 bound to ZmRAD51A1 in a dot blot experiment. These 14 sequences are found in several known maize proteins, including transcription factors. These results reveal several peptides which bind ZmRAD51A1 and support a potential role for ZmRAD51A1 in transcriptional regulation and plant defense. This study also demonstrates the applicability of phage display to basic science questions, such as the search for binding partners of a known protein, and raises the possibility of an iterated approach to test peptide sequences that closely but imperfectly align with the selected phages.