Chlamydomonas chloroplasts can use short dispersed repeats and multiple pathways to repair a double-strand break in the genome

Plant J. 2008 Mar;53(5):842-53. doi: 10.1111/j.1365-313X.2007.03376.x. Epub 2007 Nov 23.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Chlamydomonas reinhardtii / genetics*
  • Chlamydomonas reinhardtii / metabolism*
  • Chloroplasts / genetics*
  • Chloroplasts / metabolism*
  • DNA Breaks, Double-Stranded*
  • DNA Repair*
  • Gene Expression Regulation, Plant
  • Genome, Plant*
  • Introns / genetics
  • Mutation
  • Transformation, Genetic