RNase HII from Chlamydia pneumoniae discriminates mismatches incorporation into DNA-rN1-DNA/DNA duplexes
Section snippets
Materials and methods
Preparation of32P-RNA/DNA and32P-DNA-rN1-DNA/DNA duplex substrates for CpRNase HII cleavage reaction. One RNA and several DNA-rN1-DNA probes (TaKaRa, China) were artificially designed (Table 1). The probes were 5′-end labeled with [γ-32P]ATP (Yahui biotech, China) using T4 polynucleotide kinase. To prepare hybids, 5′-32P-labeled probes were annealed to 2-fold excess corresponding oligonucleotides (Table 1) in TE buffer. The hybridization mixture was heated at 95 °C for 5 min and cooled down to 20
CpRNase HII activity
To initiate functional analysis of the CpRNase HII, we purified CpRNase HII. CpRNase HII cleaves the RNA/DNA duplexes at multiple positions, but most preferably at c10–11 from the 5′-end (Fig. 1). As the typical RNA primer length in vivo varies from 7–12 ribonucleotides, the primary reaction product is consistent with a role in RNA primer removal. Studies revealed an RNase HII from B. subtilis, which showed 43% sequence identity with CpRNase HII, cleaved the same substrate at all sites, except
Acknowledgments
This work was partially supported by the National Basic Research Program of China (Grant No. 2005CB724301) and the National Science Foundation of China (Grant No. 30571012/C011003).
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