Nucleic Acids Research, Vol 24, Issue 17 3323-3331, Copyright © 1996 by Oxford University Press
F Durrenberger, AJ Thompson, DL Herrin and JD Rochaix
The mechanisms of chloroplast recombination are largely unknown. Using the
chloroplast-encoded homing endonuclease I-CreI from Chlamydomonas
reinhardtii, an experimental system is described that allows the study of
double strand break (DSB)-induced recombination in chloroplasts. The I-CreI
endonuclease is encoded by the chloroplast ribosomal group I intron of
C.reinhardtii and cleaves specifically intronless copies of the large
ribosomal RNA (23S) gene. To study DSB-induced recombination in chloroplast
DNA, the genes encoding the I-CreI endonuclease were deleted and a target
site for I-CreI, embedded in a cDNA of the 23S gene, was integrated at an
ectopic location. Endonuclease function was transiently provided by mating
the strains containing the recombination substrate to a wild-type strain.
The outcome of DSB repair was analyzed in haploid progeny of these crosses.
Interestingly, resolution of DSB repair strictly depended upon the relative
orientation of the ectopic ribosomal cDNA and the adjacent copy of the 23S
gene. Gene conversion was observed when the 23S cDNA and the neighbouring
copy of the 23S gene were in opposite orientation, leading to mobilization
of the intron to the 23S cDNA. In contrast, arrangement of the 23S cDNA in
direct repeat orientation relative to the proximal 23S gene resulted in a
deletion between the 23S cDNA and the 23S gene. These results demonstrate
that C.reinhardtii chloroplasts have an efficient system for DSB repair and
that homologous recombination is strongly stimulated by DSBs in chloroplast
DNA.
ARTICLES
Double strand break-induced recombination in Chlamydomonas reinhardtii chloroplasts
Department of Molecular Biology, University of Geneva, Switzerland.
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