Chlamydomonas chloroplasts can use short dispersed repeats and multiple pathways to repair a double-strand break in the genome.
Certain group I introns insert into intronless DNA via an endonuclease that creates a double-strand break (DSB). There are two models for intron homing in phage: synthesis-dependent strand annealing (SDSA) and double-strand break repair (DSBR). The Cr.psbA4 intron homes efficiently from a plasmid into the chloroplast psbA gene in Chlamydomonas, but little is known about the mechanism. Analysis of co-transformants selected using a spectinomycin-resistant 16S gene (16S(spec)) provided evidence for both pathways. We also examined the consequences of the donor DNA having only one-sided or no homology with the psbA gene. When there was no homology with the donor DNA, deletions of up to 5 kb involving direct repeats that flank the psbA gene were obtained. Remarkably, repeats as short as 15 bp were used for this repair, which is consistent with the single-strand annealing (SSA) pathway. When the donor had one-sided homology, the DSB in most co-transformants was repaired using two DNAs, the donor and the 16S(spec) plasmid, which, coincidentally, contained a region that is repeated upstream of psbA. DSB repair using two separate DNAs provides further evidence for the SDSA pathway. These data show that the chloroplast can repair a DSB using short dispersed repeats located proximally, distally, or even on separate molecules relative to the DSB. They also provide a rationale for the extensive repertoire of repeated sequences in this genome.
Section of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, School of Biological Sciences, University of Texas at Austin, Austin, TX 78712, USA.
Obed W
OW
Odom
Kwang-Hyun
KH
Baek
Radhika N
RN
Dani
David L
DL
Herrin
D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics* / Q000378:metabolism*; D002736:Chloroplasts / Q000235:genetics* / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D018506:Gene Expression Regulation, Plant; D018745:Genome, Plant; D007438:Introns / Q000235:genetics; D009154:Mutation; D014170:Transformation, Genetic