RNase HII from Chlamydia pneumoniae discriminates mismatches incorporation into DNA-rN1-DNA/DNA duplexes

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Abstract

It was reported that RNase HII from Chlamydia pneumoniae (CpRNase HII) had RNase H activity on RNA/DNA duplex. We have analyzed the cleavage specificity of CpRNase HII on DNA-rN1-DNA/DNA duplex (rN1, one ribonucleotide). Various mismatches were introduced into the DNA-rN1-DNA/DNA duplexes at or around the ribonucleotide. The mismatches of duplexes resulted in slower cleavage rates compared to the matched duplexes. Furthermore, a greater reduction in cleavage activity was observed for the mismatches located at or adjacent to the ribonucleotide. The mismatches at the same position of DNA-rN1-DNA/DNA duplexes have different impact on the cleavage rates of CpRNase HII depending on the types of mismatches. These findings may offer further insights into the physical binding and catalytic properties of CpRNase HII–substrate interaction.

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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