a duplex containing a (5')RNA-DNA(3') junction with one, three, or six ribonucleotides, i.e. DNA5-RNA1-DNA6/DNA12, DNA3-RNA3-DNA6/DNA12, and RNA6-DNA6/DNA12, and a substrate with a (5')DNA-RNA(3') junction, DNA5-RNA7/DNA12
RNase H2 hydrolyzes RNA of RNA/DNA hybrids and can nick duplex DNAs containing a single ribonucleotide. It shows a unique mechanism of recognition and substrate-assisted cleavage with preference for junction substrates. A conserved tyrosine residue distorts the nucleic acid at the junction, allowing the substrate to function in catalysis by participating in coordination of the active site metal ion
RNase H2 hydrolyzes RNA of RNA/DNA hybrids and can nick duplex DNAs containing a single ribonucleotide. It shows a unique mechanism of recognition and substrate-assisted cleavage with preference for junction substrates. A conserved tyrosine residue distorts the nucleic acid at the junction, allowing the substrate to function in catalysis by participating in coordination of the active site metal ion
Tma-RNase HI prefers Mg2+ to Mn2+ for activity, and specifically loses most of the Mg2+-dependent activity on removal of the hybrid binding domain and 87% of it by the mutation at the hybrid binding domain. Activity profiles of different metals and salt concentrations
the full-length and C-terminally truncated enzymes have similar activity, and both are around 600fold more active in the presence of Mn2+ compared to Mg2+, binding structure and activation mechanism, overview
the full-length and C-terminally truncated enzymes have similar activity, and both are around 600fold more active in the presence of Mn2+ compared to Mg2+, binding structure and activation mechanism, overview
the enzyme (Tma-RNase HI) and the C-terminal RNase H domain (Tma-CD) exhibit the highest activities in the presence of 1 mM MgCl2 and 0.1-5 mM MnCl2. Both proteins exhibit little activity (less than 0.01% of the maximal activity) in the presence of NiCl2, ZnCl2, CoCl2 or CaCl2. Tma-RNase HI prefers Mg2+ to Mn2+ because its maximal Mg2+-dependent activity is higher than its maximal Mn2+-dependent activity by 16fold. The enzyme specifically loses most of the Mg2+-dependent activity on removal of the hybrid binding domain and 87% of it by the mutation at the hybrid binding domain
the enzyme (Tma-RNase HI) and the C-terminal RNase H domain (Tma-CD) exhibit the highest activities in the presence of 1 mM MgCl2 and 0.1-5 mM MnCl2. Both proteins exhibit little activity (less than 0.01% of the maximal activity) in the presence of NiCl2, ZnCl2, CoCl2 or CaCl2. Tma-RNase HI prefers Mg2+ to Mn2+ because its maximal Mg2+-dependent activity is higher than its maximal Mn2+-dependent activity by 16fold
the full-length and C-terminally truncated enzymes have similar activity, and both are around 600fold more active in the presence of Mn2+ compared to Mg2+. Residue Y163 is important for binding of both (5')RNA-DNA(3') junctions and RNA/DNA substrates
the C-terminal RNase H domain loses the ability to suppress the RNase H deficiency of an Escherichia coli rnhA mutant, the hybrid binding domain is responsible for in vivo RNase H activity
Tma-RNase HI contains a hybrid binding domain at the N-terminal region. Analysis for interaction between the C-terminal and the hybrid binding domains, overview
site-directed mutagenesis, the mutation is located in the conserved GRG 2'-OH-sensing motif, the mutant has much lower activity on RNA/DNA and DNA-RNA/DNA in the presence of Mn2+. In the presence of Mg2+ it also shows reduced activity on substrates with (5')RNA-DNA(3') junction
site-directed mutagenesis, Y163 is a key residue involved in 2'-OH binding, and its mutation to phenylalanine seriously reduces activity against all tested substrates
the mutation at the hybrid binding domain does not significantly affect the structure of enzyme. The pH, salt and metal ion dependencies of the mutant enzyme are similar to those of the wild-type enzyme. Its maximal Mn2+-dependent activity is also similar to that of wild-type enzyme. However, its maximal Mg2+-dependent activity is 7.5fold lower than that of the wild-type enzyme. The binding affinity of the mutant enzyme to the substrate is 21fold higher than that of the wild-type enzyme
the C-terminal RNase H domain loses the ability to suppress the RNase H deficiency of an Escherichia coli rnhA mutant. The substrate binding affinity of Tma-RNase HI is greatly reduced on removal of the hybrid binding domain or the mutation
removal of the hybrid binding domain severely reduces the Mg2+-dependent activity of the enzyme by 750fold without significantly affecting the Mn2+-dependent activity