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.
Odom
Obed W
OW
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.
Baek
Kwang-Hyun
KH
Dani
Radhika N
RN
Herrin
David L
DL
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