Topoisomerases regulate DNA supercoiling, DNA catenation and knotting, and are important enzymes required for DNA replication, recombination and repair. Type I topoisomerases relax supercoiled DNA whereas type II topoisomerases, such as DNA gyrase, not only relax supercoiled DNA but also introduce super-coils using ATP (Singh et al. 2004). Several reports have identified type I topoisomerases in chloroplasts including a 115 kDa protein in S. oleracea (Siedlecki et al. 1983), a 54 kDa protein in Brassica oleracea (cauliflower; Fukata et al. 1991), a 69 kDa protein in P. sativum (Mukherjee et al. 1994), and a 70 kDa type IB to-poisomerase in Sinapsis alba (white mustard; Belkina et al. 2004). Evidence for type II topoisomerases in plastids include the observation that a P. sativum chloroplast transcription extract contained a gyrase-like activity which was sensitive to the gyrase inhibitor novobiocin (Lam and Chua 1987). Furthermore, an an tibody raised against yeast topoisomerase II cross-reacted with 96 and 101 kDa proteins in T. aestivum chloroplasts (Pyke et al. 1989); E. coli gyrase is comprised of 95 kDa gyrase B and 105 kDa gyrase A subunits (Reece and Maxwell 1991). A more recent proteomic study identified gyrase A- and B-like subunits in protein-nucleic acid particles from purified P. sativum chloroplasts (Phinney and Thelen 2005).
The A. thaliana nucleus contains one gene encoding a gyrase A-like subunit and three genes for gyrase B-like subunits (Wall et al. 2004). The gyrase A subunit (At3g10690) coding sequence has alternative start sites giving rise to plastid and mitochondrial targeted forms. T-DNA knockouts of the gyrase A subunit were embryo-lethal. One gyrase B subunit appeared to be targeted to plastids, the second to mitochondria, whereas the location of the third was unclear but was possibly located in the nucleus/cytosol (Wall et al. 2004). Knockouts of either organ-elle-targeted gyrase B subunit were seedling-lethal rather than the more severe embryo-lethal phenotype of gyrase A mutants. This suggested the gyrase B sub-units complement each other to a limited extent indicating their products might be targeted to both organelles (Wall et al. 2004). This appears to be the case. An alternative upstream non-AUG start codon (most probably CUG) in one gyrase B subunit (gene At3g10270) gives rise to an N-terminus that confers dual-targeting to mitochondria and plastids. Alternative translation start sites in the coding sequence for the second organelle-targeted gyrase B subunit (gene At5g04130) give rise to either mitochondrial or plastid targeted proteins (Christensen et al. 2005). In summary, one gyrase A subunit and two gyrase B subunits appear to be targeted to both plastids and mitochondria in A. thaliana (Table 2). Presequences that confer dual-targeting of gyrase A and B subunits to mitochondria and chloroplasts have been found in Nicotiana benthamiana (Cho et al. 2004).
The effects of transient downregulation of organelle-targeted gyrase A and B subunit expression were studied in N. benthamiana by virus-induced gene silencing (Cho et al. 2004). Downregulation of gyrase A or B subunits prevents chloro-plast development giving rise to white or yellow leaf sectors. Larger nucleoids and a mixture of heterogeneous high MW DNA molecules in plastids, possibly representing tangled DNA and their breakage products, are consistent with a crucial role for gyrase in untangling plastid DNA following replication and recombination (Cho et al. 2004). A role for gyrase in plastid DNA maintenance is supported by an earlier study where the gyrase inhibitors novobiocin and naladixic acid were shown to reduce the copy number of plastid DNA in Solanum nigrum suspension cultures (Ye and Sayre 1990).
In addition to roles in DNA-RRR pathways gyrase activity can also influence transcription (Chapter 5) through changes in supercoiling (Reece and Maxwell 1991). Mutations in gyrase activity might therefore impact on plastid gene expression as well as genome maintenance. The gyrase inhibitors novobiocin and naladixic acid were found to alter the accumulation of plastid transcripts in C. reinhardtii (Thompson and Mosig 1985). Addition of novobiocin to a P. sativum chloroplast transcription system containing cloned plastid genes inhibited the expression of the atpB gene to a larger extent than the rbcL gene. This raised the possibility that template topology may enable differential regulation of plastid genes (Lam and Chua 1987).
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