期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 107, 期 31, 页码 13954-13959出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1004326107
关键词
DNA repair; evolution; homing endonuclease; I-CreII; plastid DNA
资金
- Department of Energy [DE-FG03-02ER15352]
- Welch Grant [F-1164]
- Texas-Advanced Research Program [003658-0144-2007]
- National Science Foundation [IOS-0822811, IOS-0849287]
- Division Of Integrative Organismal Systems
- Direct For Biological Sciences [0822811, 0849287] Funding Source: National Science Foundation
Chloroplast DNA (cpDNA) is under great photooxidative stress, yet its evolution is very conservative compared with nuclear or mitochondrial genomes. It can be expected that DNA repair mechanisms play important roles in cpDNA survival and evolution, but they are poorly understood. To gain insight into how the most severe form of DNA damage, a double-strand break (DSB), is repaired, we have developed an inducible system in Arabidopsis that employs a psbA intron endonuclease from Chlamydomonas, I-CreII, that is targeted to the chloroplast using the rbcS1 transit peptide. In Chlamydomonas, an I-CreII-induced DSB in psbA was repaired, in the absence of the intron, by homologous recombination between repeated sequences (20-60 bp) abundant in that genome; Arabidopsis cpDNA is very repeat poor, however. Phenotypically strong and weak transgenic lines were examined and shown to correlate with I-CreII expression levels. Southern blot hybridizations indicated a substantial loss of DNA at the psbA locus, but not cpDNA as a whole, in the strongly expressing line. PCR analysis identified deletions nested around the I-CreII cleavage site indicative of DSB repair using microhomology (6-12 bp perfect repeats, or 10-16 bp with mismatches) and no homology. These results provide evidence of alternative DSB repair pathways in the Arabidopsis chloroplast that resemble the nuclear, microhomology-mediated and nonhomologous end joining pathways, in terms of the homology requirement. Moreover, when taken together with the results from Chlamydomonas, the data suggest an evolutionary relationship may exist between the repeat structure of the genome and the organelle's ability to repair broken chromosomes.
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