Plastid localised RecA

The RecA protein promotes strand transfer and heteroduplex formation between DNA duplexes in prokaryotes (CameriniOtero and Hsieh 1995). RecA is required for DNA replication, recombination and repair pathways (Kowalczykowski 2000). The central role of RecA protein in homologous recombination is illustrated by its position in the double-strand break recombination model (Szostak et al. 1983) in Figure 15. Absence of recA function reduces the frequency of homologous recombination by 10,000-fold in E. coli (CameriniOtero and Hsieh 1995). Plastids contain a homologue of RecA, which was first identified as a 39 kDa protein in P. sa-tivum chloroplasts using polyclonal antibodies against E. coli RecA protein

(Cerutti et al. 1992). This provided the first evidence for a RecA-mediated homologous recombination pathway in plastids. Accumulation of the P. sativum RecA-like protein appears to increase following exposure of protoplasts to DNA damaging agents (Cerutti et al. 1993). The presence of a RecA homologue in P. sativum chloroplasts has been recently confirmed by proteomics of protein-nucleic acid particles from purified chloroplasts (Phinney and Thelen 2005). The A. thaliana nuclear genome contains four genes encoding RecA-like proteins related to the bacterial RecA proteins (Khazi et al. 2003). Two of these genes encode or-ganelle-targeted proteins (Khazi et al. 2003). Gene At1g79050 encodes a 439 amino acid protein (Table 2) that is predicted to be targeted to chloroplasts (Tar-getP score 0.841; Emanuelsson et al. 2000) and is imported into isolated P. sativum chloroplasts (Cao et al. 1997), whereas gene At3g10140 encodes a 389 amino acid protein that is targeted to mitochondria (Khazi et al. 2003). Both RecA-like proteins share 36% identity excluding N- and C-terminal extensions (Khazi et al. 2003). The At3g10140 mitochondrial RecA-like protein partially complements an E. coli recA mutant and provides tolerance to the methyl methane sulfonate (MMS) and mitomycin C (Khazi et al. 2003), which are DNA damaging agents.

C. reinhardtii plastids contain a RecA homologue (Nakazato et al. 2003). The influence of altered RecA activity on plastid recombination was addressed by expressing E. coli WT and dominant negative RecA proteins in C. reinhardtii chloroplasts (Cerutti et al. 1995). Expression of WT E. coli RecA in C. reinhardtii chloroplasts increased the frequency of homologous recombination between 216 bp direct repeats by over 15-fold. This indicates that recombination between direct repeats appears to be limited by RecA-mediated strand exchange. In contrast, overexpressed WT E. coli RecA did not enhance survival of cells exposed to DNA damaging agents indicating that chloroplast DNA repair pathways are not limited by RecA activity but by processing of DNA substrates generated by DNA damaging agents. An E. coli dominant-negative RecA protein reduced recombination between direct repeats and DNA repair in chloroplasts consistent with a negative influence on a RecA-mediated DNA-RRR pathway in plastids (Cerutti et al. 1995).

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