Functional Research Of Rnase Hs From Hlamydophila Pneumoniae

RNase Hs can cleave the RNA portion of RNA/DNA hybrids orDNA-RNA-DNA/DNA chimeric substrate. Based on amino acidsequences and structural similarities, RNase Hs have been classified intotype1and type2RNase H, and prokaryotic RNase Hs are divided intothree groups, RNase HI, HII and HIII. RNase HI is a type1RNase H,while RNase HII and HIII are both of type2.Accumulated sequence data have indicated that RNase HIIs arepresent in nearly all sequenced bacteria. However, many bacterial speciestypically contain either RNase HI or HIII but not both. Although allRNase Hs efficiently cleave DNA-rNn-DNA/DNA dsDNA （ds,double-stranded, n≥4） or RNA/DNA hybrids, the DNA-rN₁-DNA/DNAsubstrate is nicked only by RNase HIIs （or H2s in eukaryotes） at the5′-side of the ribonucleotide.Chlamydophila pneumoniae, a pathogenic eubacterial intracellularparasite for humans and some animals, infects the mucosal surfaces of therespiratory tract, causing pharyngitis, bronchitis and pneumonia.Complete genome sequence revealed that two RNase Hs of type2exist inC. pneumoniae AR39, i.e., CpRNase HII and CpRNase HIII. Ourprevious researches indicated that both CpRNase HII and HIII haveRNase H activities and CpRNase HII is able to cleave theDNA-rN₁-DNA/DNA substrate in the presence of Mg2+. In the presentstudy, our results showed purified CpRNase HIII was able to efficiently process DR1D-Iin the presence of Mn²⁺, with maximal cleavage observedat0.1mM Mn²⁺. This is the first report on the cleavage ability of RNaseHIII to DNA-rN₁-DNA/DNA hybrids.Our biochemical results indicated that in a Mn²⁺-rich environment,CpRNase HII activity was strongly inhibited, whereas CpRNase HIII wasable to cleave the DNA-rN₁-DNA/DNA substrate, both CpRNase Hswere sensitive to the fluctuation of metal ions. However, when bothCpRNase Hs were present, the loss of the activity in one enzyme could becompensated by the other, thus, the negative effects of the metal ions ontotal RNase H activity were negligible. On the other hand, whenRNA-DNA/DNA substrates or RNA/DNA hybrids were used assubstrates, both CpRNase HII and HIII tolerated the fluctuations ofdivalent ions.To evaluate the enzymatic activities and characteristics of CpRNaseHII and HIII in vivo, we constructed three E. coli rnh knockout strainswith different genotypes: LZ1[DY329, ΔrnhA ΔrnhB:: CprnhB], LZ2[DY329, ΔrnhA:: CprnhC ΔrnhB:: CprnhB], and LZ3[DY329, ΔrnhA::CprnhC ΔrnhB]. The CprnhB and CprnhC genes encode CpRNase HIIand CpRNase HIII of C. pneumoniae AR39, respectively; the rnhA andrnhB genes encode E. coli RNase HI and HII, respectively. Our resultsdemonstrated that in the presence of Mn²⁺, CpRNase HIII couldcomplement the E. coli rnh knockout mutant and undertake all RNase Hactivities in E. coli; however, the complementary ability of CpRNase HIIneeded Mg2+and was inhibited by Mn²⁺. Limited RNase H activitysignificantly restricted E. coli growth. However, the cleavage ofRNA/DNA hybrids and Okazaki-fragment substrates by CpRNase Hs invitro was not obviously influenced by the flux of metal ions, suggesting the in vivo processing of these two physiological substrates were notaffected under the different culture conditions investigated in this study.Therefore, it seemed likely that the negative effect on the bacterial growthwas a result of the limited ability to remove chimeric ribonucleotidesfrom double-stranded DNA （dsDNA）. The results of thealkaline-sensitivity assays further supported this hypothesis: the genomesof RNase H-deficient strains were very sensitive to alkali.The intracellular Mn²⁺concentrations of the mutant strains weremeasured with inductively coupled plasma atomic emission spectrometry（ICP-AES）, and the results showed when Mn²⁺was added into the growthmedium, the intracellular concentrations of Mn²⁺changed to facilitate thefunction of CpRNase HIII but inhibited CpRNase HII activity.Furthermore, real-time PCR results confirmed that the in vivo expressionof CprnhB and CprnhC （the encoding genes of CpRNase Hs） in mutantstrains was stable regardless of the presence or absence of Mn²⁺. Theseresults demonstrated the growth defects of the constructed E. coli mutantstrains were due to the cleavage deficiency of CpRNase Hs for the DNA-rN₁-DNA/DNA substrate.Our results indicate that the in vivo relationship and functions of twoCpRNase Hs are cooperative and complementary. RNase HIII is not onlyable to a substitute for RNase HI but is also a conditional replacement forRNase HII. Therefore, CpRNase HIII is perceived to be a necessaryRNase H in C. pneumoniae and is not simply a redundant factor.After clarifying the fact that CpRNase HIII can cleaveDNA-rN₁-DNA/DNA substrate, we further examined thestructure-function relationship of this protein. For this purpose,mutagenesis analyses and molecular dynamics simulations （MD） were performed on CpRNase HIII. Our results elucidated the mechanism ofribonucleotide recognition employed by CpRNase HIII, indicating thatthe G95K96G97motif of CpRNase HIII functioned as the main surfaceinteracting with ribonucleotides in a similar way to the GR （K） G motif ofRNase HIIs. However, CpRNase HIII lacks tyrosine, which is necessaryfor RNase HII to recognize the chimeric single ribonucleotide in dsDNA.Interestingly, our MD results showed that the Ser94of CpRNase HIIIformed a stable hydrogen bond with the deoxyribonucleotide at the （5’）RNA-DNA （3’） junction, moving this nucleotide away from the chimericribonucleotide. This movement apparently deformed the nucleic acidbackbone at the RNA-DNA junction, and allowed the ribonucleotide tobe touched accurately. Following the inferences drawn from MD,biochemical results indicated that Ser94was necessary for the catalyticactivity on DNA-rN₁-DNA/DNA substrate. Our results may helpelucidate the distinct substrate recognitions of different RNase Hs and is anecessary addition to the RNase H field.