Precise genetic engineering for crop development is an important commercial goal for the plant biotechnology industry. One major impediment for gene targeting by homologous recombination in plants is competition by enzymes of the nonhomologous end joining pathway (NHEJ) for the introduced DNA. Recent studies in yeast and mammals have revealed the central roles of DNA ligase IV and XRCC4 proteins in NHEJ. DNA ligase4 and XRCC4 directly interact in two hybrid assays. A separate study has shown the importance of NHEJ pathway for T-DNA integration in yeast when the T-DNA is transferred from Agrobacteria into yeast. Our hypothesis is that if NHEJ pathway is transiently inhibited during T-DNA infection or during transformation with linear DNA by particle gun bombardment, more DNA will be able to interact with plant chromosomal DNA because competition with NHEJ will be less. This should increase the frequency of homologous recombination and gene targeting. Towards testing this hypothesis, we are setting up a yeast based NHEJ assay dependent on plant DNA ligase IV and plant XRCC4, and a direct interaction assay. We are screening a peptide aptamer library in yeast to find aptamer hits that inhibit the direct interaction. Then we will use the NHEJ assay to find among the hits those that inhibit NHEJ. Ultimately, these aptamers will be transiently expressed in plant cells during T-DNA or other double stranded DNA delivery to inhibit NHEJ and thus to stimulate homologous gene targeting.
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