Biochemical characterization and functional complementation of ribonuclease HII and ribonuclease HIII from Chlamydophila pneumoniae AR39

摘要

Chlamydophila pneumoniae AR39 contains two different ORFs (CP0654 and CP0782) encoding ribonuclease H (RNase H) homologues, Cpn-RNase HII and Cpn-RNase HIII. Sequence alignments show that the two homologues both contain the conserved motifs of type 2 RNase H, and Cpn-RNase HII has the conserved active-site motif (DEDD) of RNase HII. Cpn-RNase HIII also contains a unique active-site motif (DEDE), common to other RNase HIIIs. Complementation assays indicated that Cpn-RNase HII can complement both Escherichia coli RNase HII and RNase HI, but Cpn-RNase HIII can only complement the latter. In vitro enzyme activity experiments showed that neither Cpn-RNase HII nor Cpn-RNase HIII is thermostable and their optimum pH values were 9.0 and 10.0, respectively. Cpn-RNase HII cleaves a 12?bp RNA–DNA substrate at multiple sites, but Cpn-RNase HIII at only one site. When a 35?bp DNA–RNA–DNA/DNA chimeric substrate was used, cleavage was only observed with Cpn-RNase HII. These results indicate that the RNase H combination of C. pneumoniae AR39 is not simple substitution of E. coli RNase H, perhaps representing a more primordial type. This is believed to be the first in vivo functional study of Chlamydophila RNase Hs and the results should cntribute to the analysis of RNase Hs of other parasite species.