The DNA unwinding steps of DNA-RRR pathways include the generation of single-stranded recombination substrates (Fig. 15) and are carried out by ATP-dependent helicases. In an early study, a helicase fraction containing 6-8 protein bands was purified from G. max chloroplasts and shown to remove a 28 base oli-gomer from a single-stranded circular M13 template (Cannon and Heinhorst 1990). A similar biochemical approach identified 68 and 78 kDa helicases in purified P. sativum chloroplasts (Tuteja 2003). The 78 kDa helicase was stimulated by DNA fork structures indicating a role in replication (Tuteja and Phan 1998). Unwinding was inhibited by nogalamycin and ATPase activity by daunorubicin (Tuteja and Phan 1998). Both nogalamycin and daunorubicin are major groove intercalating agents. Chloroplast helicases appear to be sensitive to actinomycin C1 and resistant to ellipticine whereas the converse is true of nuclear helicases (Tuteja 2003).
A genomics based study has identified two O. sativa RecQ helicase homologues that are likely to be present in plastids (Saotome et al. 2006). Transient expression of GFP-fusions in onion epidermal cells indicated one 588 amino acid RecQ-like protein (OsRecQ1, Nucleotide Acc No. AK101124) was targeted to nuclei and plastids, whereas the second 844 amino acid Rec Q-like protein (OsRec-Qsim, Nucleotide Acc No. AK072977) was targeted predominantly to plastids. RNA levels for these RecQ-like proteins appeared highest in meristematic tissues containing immature plastids and did not increase in response to light and chloro-plast development. A role for these proteins in repair was suggested by the observations that RNA encoding the 588 amino acid RecQ-like protein increased in levels in response to the four DNA damaging agents, mitomycin C, H2O2, MMS and bleomycin. Expression of the RNA encoding the 844 amino acid protein increased following treatment with mitomycin C and bleomycin and increased slightly with MMS (Saotome et al. 2006).
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