Studies of site-specific DNA double strand break repair in plants

<p>The maize transposon <i>Ac/Ds</i> transposes by a “cut and paste"” mechanism, leaving a site-specific DSB at the <i> Ds </i> locus for repair. Previous studies in maize and Arabidopsis showed that <i>Ac/Ds</i> excision can stimulate homologous recombination between tandem duplicated repeats and between ectopic homologies. In this study, we tested the efficacy of such <i>Ac/Ds</i> excision-induced homologous recombination to achieve gene targeting in Arabidopsis. A defective visual-selective dual marker harboring <i> Ds </i> element is transformed into Arabidopsis as target locus, and donor homology is provided by Agrobacterium-mediated T-DNA by floral dipping. The DSB inducer <i> Ac </i> is either first crossed into target lines or co-transformed with donor T-DNA, and several independent germinal recombinants have been recovered from both strategies with gene targeting frequency of 0.3-2.0X10^(-3). We also attempted gene targeting by ectopic recombination, with donor homology provided by the concomitant integrated T-DNA copies in genome. With this approach the gene targeting frequency turns out less than 6.7X10^(-6). Our results suggest that <i>Ac/Ds</i> transposon-induced homologous recombination may provide an alternative gene targeting strategy. The implications of these results for plant gene targeting are discussed.</p> <p>Multiple pathways are used to repair Double Strand Breaks (DSBs), including Nonhomologous End Joining (NHEJ) and Homologous Recombination (HR) which can take Single Strand Annealing (SSA) or Gene Conversion (GC) pathway. DSB repair in plants has been extensively studied using site-specific DSB agents, including two mechanistically different inducers transposon <i>Ac/Ds</i> and endonuclease <i>I-SceI</i>. A direct comparison between <i>Ac/Ds</i> and <i>I-SceI</i> in DSB repair, however, is lacking due to the diversity of systems used among previous studies. In addition, only a few DSB repair studies addressed germinal recombination frequencies. In this study, we developed three constructs (HRS1, 2 and 3) that allow comparison of multiple pathways for repair of DSBs induced by <i>Ac/Ds</i> excision and by <i>I-SceI</i> cutting at the same chromosomal loci. The results show that differential pathway utilization exists between the repair of <i>Ac/Ds</i> excision and <i>I-SceI</i> induced DSBs: (i) In somatic tissues, <i>Ac/Ds</i> induced HR preferentially utilizes SSA and/or represses GC 4 fold higher than <i>I-SceI</i>; (ii) In germinal tissues, repair of <i>Ac/Ds</i> induced DSBs favors NHEJ and strongly represses HR by 2 to 3 orders magnitude, whereas <i>I-SceI</i>-induced DSBs are repaired equally by NHEJ and HR; (iii) Furthermore, <i>Ac/Ds</i> induced germinal HR preferentially utilizes SSA and/or represses GC 5 fold higher than <i>I-SceI</i>; and (iv) germinal tissues preferentially utilize SSA 3 fold higher than GC compared to somatic tissues for both <i>Ac/Ds</i> and <i>I-SceI</i> induced HR. Despite these inequalities, a roughly positive correlation exists between somatic and germinal HR frequencies for both <i>Ac/Ds</i> and <i>I-SceI</i>-induced DSBs. Overall, DSB repair pathway and frequency is strongly affected by both cell type (somatic vs. germinal) and DSB agent. The striking difference of repair pathway utilization between <i>Ac/Ds</i> and <i>I-SceI</i> suggests specific role(s) of <i>Ac/Ds</i> in DSB repair. The hairpin intermediate generated prior to DSB formation, or <i> Ac </i> transposase <i>per se</i>, may promote NHEJ and SSA and/or repress GC. These results provide new insight into how transposons affect genome structure and also shed light on the biology of DSB-induced HR that may facilitate the development of genome modification tools for plants.</p>

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

dissertation

Department dc:contributor.department

Department of Genetics, Development, and Cell Biology (LAS)

Year dc:date.issued

2010

Author dc:creator

• Kuang, Xianyan

Advisor dc:contributor.advisor

• Thomas A. Peterson

Language dc:language.iso

en

DOI

https://doi.org/10.31274/etd-180810-2459

Identifier

archive/lib.dr.iastate.edu/etd/11350/

OAI identifier oai:identifier

oai:dr.lib.iastate.edu:20.500.12876/25556