Open AccessNonhomologous DNA End-Joining in Plants: Genes and Mechanisms
Author(s) -
Clifford F. Weil,
Anne Britt,
Avraham A. Levy
Publication year - 2001
Language(s) - English
Resource type - Reports
DOI - 10.32747/2001.7585194.bard
Subject(s) - ku80 , biology , dna repair , genetics , gene knockout , homologous recombination , gene , ku70 , reverse genetics , arabidopsis , non homologous end joining , plasmid , dna ligase , gene targeting , chromatin , mutant , transcription factor , dna binding protein
Repair of DNA breaks is an essential function in plant cells as well as a crucial step in addition of modified DNA to plant cells. In addition, our inability to introduce modified DNA to its appropriate locus in the plant genome remains an important hurdle in genetically engineering crop species.We have taken a combined forward and reverse genetics approach to examining DNA double strand break repair in plants, focusing primarily on nonhomologous DNA end-joining. The forward approach utilizes a gamma-plantlet assay (miniature plants that are metabolically active but do not undergo cell division, due to cell cycle arrest) and has resulted in identification of five Arabidopsis mutants, including a new one defective in the homolog of the yeast RAD10 gene. The reverse genetics approach has identified knockouts of the Arabidopsis homologs for Ku80, DNA ligase 4 and Rad54 (one gene in what proves to be a gene family involved in DNA repair as well as chromatin remodeling and gene silencing)). All these mutants have phenotypic defects in DNA repair but are otherwise healthy and fertile. Additional PCR based screens are in progress to find knockouts of Ku70, Rad50, and Mre11, among others. Two DNA end-joining assays have been developed to further our screens and our ability to test candidate genes. One of these involves recovering linearized plasmids that have been added to and then rejoined in plant cells; plasmids are either recovered directly or transformed into E. coli and recovered. The products recovered from various mutant lines are then compared. The other assay involves using plant transposon excision to create DNA breaks in yeast cells and then uses the yeast cell as a system to examine those genes involved in the repair and to screen plant genes that might be involved as well. This award supported three graduate students, one in Israel and two in the U.S., as well as a technician in the U.S., and is ultimately expected to result directly in five publications and one Masters thesis.