Supporting information
Junction ribonuclease activity specified in RNases HII/2
Naoto Ohtani*, Masaru Tomita, and Mitsuhiro Itaya
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan
1
Fig. S1. Time course of the cleavage of the 12 bp RNA-DNA/DNA substrate.
The 12 bp RNA-DNA/DNA hybrid, in which the 3′-end of the RNA-DNA strand was
fluorescein-labeled, was incubated at 37oC with each protein in the presence of 5 mM
MnCl2 (A) or MgCl2 (B): Eco-HI, 0.25 nM for MnCl2 and 25 nM for MgCl2 of
Eco-RNase HI; Eco-HII, 2.5 M of Eco-RNase HII; Bsu-HIII, 25 nM of Bsu-RNase
HIII; Tth-J, 0.25 M of Tth-JRNase. The concentration of the substrate was 0.5 M.
Products were separated on a 20% polyacrylamide gel containing 7 M urea as described
in Experimental procedures. M as a marker represents 7 b RNA1-DNA as shown in
Table 1. Lanes 1-8 represent samples incubated with each amount of the protein for 0, 1,
3, 5, 10, 15, 20, and 30 min. The cleavage site is shown schematically at the bottom.
The asterisk indicates the labeled site, and lowercase and uppercase letters represent
RNA and DNA, respectively.
2
Appendix S1.
Supplementary experimental Procedures
Enzyme preparations—A plasmid for the N-terminal His-tagged Aae-RNase
HIII was constructed by ligating the DNA fragment containing the A. aeolicus rnhC
gene, which was chemically synthesized by TaKaRa Bio (Shiga, Japan), to the
NdeI-BamHI site of pET-28b. On the other hand, plasmids for the N-terminal
His-tagged Mmo- and Mpn-RNases HIII were constructed using methods similar to
those described for Bsu-RNase HIII. Genomic DNA of M. mobile 163K or M.
pneumoniae M129 was used as a template for PCR. The primer sequences to amplify
the
MMOB1230
gene
encoding
Mmo-RNase
HIII
were
5'-GCGCGCGCGCATATGAATTTCAATGAATATGTAACAATT-3′ for forward primer
and 5'-CGCGCGCTCGAGTTATTATGCTTTTTTAAGTCCAAAAAG-3′ for reverse
primer, and those to amplify the MPN118 gene encoding Mpn-RNase HIII were
5'-GCGCGCGCGCATATGCAACACGCAAACACAACCGCACTTT-3′ for forward
primer and 5'-CGCGCGCTCGAGTTATTAAGCCAGCTGCTTTAGGAAACTA-3′ for
reverse primer, where underlined bases show the positions of the NdeI (forward primer)
and XhoI (reverse primer) sites. Overproduction in Rosetta(DE3) and purification were
carried out as described for Bsu-RNase HIII. The purified protein was dialyzed against
20 mM Tris-HCl (pH 8.0) containing 1 mM EDTA and 0.5 M NaCl for Mmo-, and
Mpn-RNases HIII, or 20 mM NaOAc (pH 5.5) containing 0.1 M NaCl for Aae-RNase
HIII, concentrated, and used for further analyses. The protein concentration was
determined by measuring UV absorption using A280 values of 0.1% solution of 0.86,
0.50, and 0.44 for the His-tagged Aae-, Mmo-, and Mpn-RNases HIII, which were
calculated from  values of 1576 M-1cm-1 for Tyr and 5225 M-1cm-1 for Trp at 280 nm.
3
Appendix S2. Examination of RNases HIII for JRNase activity.
Despite sequence similarity to RNase HII/2 enzymes, Bsu-RNase HIII exhibited
no JRNase activity. Therefore, to clarify whether or not other RNases HIII, as RNase
HII paralogs, show JRNase activity, RNases HIII from Aquifex aeolicus, Mycoplasma
mobile 163K, and Mycoplasma pneumoniae M129 (Aae-, Mmo-, and Mpn-RNases HIII,
respectively) were also examined. Aae-, Mmo-, and Mpn-RNases HIII are encoded by
the rnhC [1-3], the MMOB1230 [4], and the MPN118 [2,5,6] genes, respectively. The
enzyme preparations are shown in Fig. S2A. Three RNases HIII in addition to
Bsu-RNase HIII were active as the RNase H enzyme (Fig. S2B). Bsu-, Mmo-, and
Mpn-RNases HIII slightly preferred the Mg2+ ion to the Mn2+ ion for RNase H activity,
whereas Aae-RNase HIII clearly preferred the Mn2+ ion. When the 12 bp
RNA-DNA/DNA heteroduplex was used as a substrate, Bsu-, and Mmo-RNases HIII
cleaved the 12 bp RNA-DNA/DNA hybrid at the 5′ side of the last ribonucleotide at the
RNA-DNA junction (Fig. S2C). However, none of these RNase HIII enzymes cleaved
the 12 bp RNA-DNA/RNA heteroduplex at any sites in the presence of 5 mM MnCl2
(Fig. S2D) or MgCl2 (data not shown). Therefore, the absence of JRNase activity is
likely a common feature among RNase HIII enzymes.
References
1.
Ohtani N, Haruki M, Morikawa M, Crouch RJ, Itaya M & Kanaya S (1999)
Identification of the genes encoding Mn2+-dependent RNase HII and
Mg2+-dependent RNase HIII from Bacillus subtilis: classification of RNases H
into three families. Biochemistry 38, 605-618.
2.
Ohtani N, Haruki M, Morikawa M & Kanaya S (1999) Molecular diversities
of RNases H. J Biosci Bioeng 88, 12-19.
3.
Deckert G, Warren PV, Gaasterland T, Young WG, Lenox AL, Graham DE,
Overbeek R, Snead MA, Keller M, Aujay M, Huber R, Feldman RA, Short JM,
Olsen GJ & Swanson RV (1998) The complete genome of the
hyperthermophilic bacterium Aquifex aeolicus. Nature 392, 353-358.
4.
Jaffe JD, Stange-Thomann N, Smith C, DeCaprio D, Fisher S, Butler J, Calvo
S, Elkins T, FitzGerald MG, Hafez N, Kodira CD, Major J, Wang S,
Wilkinson J, Nicol R, Nusbaum C, Birren B, Berg HC & Church GM (2004)
The complete genome and proteome of Mycoplasma mobile. Genome Res 14,
4
1447-1461.
5.
Bellgard MI & Gojobori T (1999) Identification of a ribonuclease H gene in
both Mycoplasma genitalium and Mycoplasma pneumoniae by a new method
for exhaustive identification of ORFs in the complete genome sequences.
FEBS Lett 445, 6-8.
6.
Dandekar T, Huynen M, Regula JT, Ueberle B, Zimmermann CU, Andrade
MA, Doerks T, Sánchez-Pulido L, Snel B, Suyama M, Yuan YP, Herrmann R
& Bork P (2000) Re-annotating the Mycoplasma pneumoniae genome
sequence: adding value, function and reading frames. Nucleic Acids Res 28,
3278-3288.
5
Fig. S2A. SDS-PAGE of the purified RNases HIII.
Approximately 250 pmol of each enzyme was subjected to 12% SDS-PAGE and stained
with Coomassie Brilliant Blue: M, a molecular mass marker from Bio-Rad; lane 1,
Bsu-RNase HIII; lane 2, Aae-RNase HIII; lane 3, Mmo-RNase HIII; lane 4, Mpn-RNase
HIII. Molecular mass standards are indicated on the left-hand side of the gel.
Fig. S2B. RNase H activities of RNases HIII.
The 12 bp RNA/DNA hybrid, in which the 5′-end of the RNA strand was
fluorescein-labeled, was incubated at 37oC for 15 min with each protein in the presence
of 5 mM MnCl2 (i) or MgCl2 (ii); Bsu-HIII, Bsu-RNase HIII; Aae-HIII, Aae-RNase
HIII; Mmo-HIII, Mmo-RNase HIII; Mpn-HIII, Mpn-RNase HIII. The concentration of
the substrate was 0.5 M. Products were separated on a 20% polyacrylamide gel
containing 7 M urea as described in Experimental procedures. M as a marker represents
products resulting from partial digestion of the 12 b RNA with snake venom
6
phosphodiesterase (VPDase). Lane 1 represents samples without the enzyme, whereas
lanes 3-6 represent samples incubated with each amount of the protein (2.5 nM, 25 nM,
0.25 M, and 2.5 M, respectively). Since the lanes are numbered in a similar manner
to those in Fig. 2, lane 2 is not in this Figure.
Fig. S2C. Cleavages of the 12 bp RNA-DNA/DNA substrate.
The 12 bp RNA-DNA/DNA hybrid, in which the 3′-end of the RNA-DNA strand was
fluorescein-labeled, was incubated at 37oC for 15 min with each protein in the presence
of 5 mM MnCl2 (i) or MgCl2 (ii). M as a marker represents 7 b RNA1-DNA as shown in
Table 1. Lane representation is similar to that shown in Fig. S2B. The cleavage site is
shown schematically at the bottom. The asterisk indicates the labeled site, and
lowercase and uppercase letters represent RNA and DNA, respectively.
Fig. S2D. JRNase activities on the 12 bp RNA-DNA/RNA substrate.
The 12 bp RNA-DNA/RNA hybrid, in which the 3′-end of the RNA-DNA strand was
labeled, was incubated in the presence of 5 mM MnCl2. Lanes and schemata are
7
represented as shown in Figs. S2B and C.
8