Cleavage of Single Strand RNA Adjacent to RNA-DNA Duplex Regions by Escherichia coli RNase H1

Abstract

RNase H1 from Escherichia coli cleaves single strand RNA extending 3 from an RNA-DNA duplex. Substrates consisting of a 25-mer RNA annealed to complementary DNA ranging in length from 9 -17 nucleotides were designed to create overhanging single strand RNA regions extending 5 and 3 from the RNA-DNA duplex. Digestion of single strand RNA was observed exclusively within the 3 overhang region and not the 5 overhang region. RNase H digestion of the 3 overhang region resulted in digestion products with 5-phosphate and 3-hydroxyl termini. The number of single strand RNA residues cleaved by RNase H is influenced by the sequence of the single strand RNA immediately adjacent to the RNA-DNA duplex and appears to be a function of the stacking properties of the RNA residues adjacent to the RNA-DNA duplex. RNase H digestion of the 3 overhang region was not observed for a substrate that contained a 2-methoxy antisense strand. The introduction of 3 deoxynucleotides at the 5 terminus of the 2-methoxy antisense oligonucleotide resulted in cleavage. These results offer additional insights into the binding directionality of RNase H with respect to the heteroduplex substrate.RNase H hydrolyzes RNA in an RNA-DNA duplex (1). RNase H activity appears to be ubiquitous in eukaryotes and bacteria (2, 3). This family of enzymes functions as endonucleases exhibiting limited sequence specificity and requiring divalent cations to produce 5Ј-phosphate and 3Ј-hydroxyl termini (4).RNase H1 from Escherichia coli is perhaps the best characterized isotype of this family of enzymes. The substrate requirements for RNase H1 have been studied extensively. The dissociation constants for various duplexes and E. coli RNase H1 indicate that this enzyme is a double strand RNA-binding protein that cleaves RNA only in an RNA-DNA duplex (5). Furthermore, the cleavage specificity of the enzyme appears to be due to the catalytic process and not the binding interaction between RNase H and the substrate duplex. For example, although RNase H exhibited a binding affinity for substrates containing 2Ј-methoxy modifications approximately equal to the unmodified RNA-DNA heteroduplex, RNase H activity was ablated by these modifications. Kinetic analyses of substrates containing RNA duplexed with chimeric oligonucleotides composed of a mixture of deoxy-and 2Ј-modified nucleotides demonstrated that the rate of cleavage was a function of the length of the deoxy portion of the chimeric oligonucleotide, i.e. the cleavage rate decreased with a diminishing number of contiguous deoxynucleotides, and complete loss in activity was observed for chimeric oligonucleotides containing less than 4 contiguous deoxynucleotides (6, 7). In all cases E. coli RNase H1 cleavage of the RNA was observed to occur exclusively within regions base-paired to DNA.In this communication we report that E. coli RNase H1 can cleave single strand RNA extending 3Ј from an RNA-DNA duplex. The fact that the products are 5Ј-phosphate and 3Ј-hydroxyl suggests that the enzyme cleaves single and double strand regi...

Properties of Cloned and Expressed Human RNase H1

“…The smallest "gap-mer" for which cleavage could be measured was a 5 deoxynucleotide gap. These data are highly consistent with observations we have previously reported for E. coli RNase H1, except that for the bacterial enzyme, the minimum gap size was 4 deoxynucleotides (18,20,21).…”

“…A duplex containing a phosphodiester oligodeoxynucleotide hybridized to a phosphodiester 2Ј-methoxy oligonucleotide as the noncleavable substrate is considered most like DNA-RNA. Table IV shows the results of these studies and compares them to previously reported results for the E. coli enzyme performed under similar conditions (20,21). Clearly, the affinity of the human enzyme for its DNA-RNA like substrate (DNA-2Ј-methoxy) was substantially greater than that of the E. coli enzyme, consistent with the differences observed in K m (Table III).…”

Ribonuclease H attack of leukaemic fused transcripts AML1‐MTG8(ETO) by DNA/RNA chimeric hammerhead ribozymes

“…The digestion appeared to be endonucleolytic at preferential sites, which is compatible with the reported properties of human RNase H (Frank et al 1994). Furthermore, the digestion seemed to occur at the outside of the RNA/DNA hybrid region, the same phenomenon as was reported for E. coli RNase H (Lima & Crooke 1997).…”

Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase

“…The RT-ROL method identified extendible sites in the loop region between nt 18-25 in the ha-ras RNA stemloop (Fig+ 5A), as shown by the analysis of RT-ROL products separated by gel electrophoresis (Fig+ 5B)+ The RNase H assay for the ha-ras RNA fragment identified accessible sites between nt 20-27+ This region is of similar size to that identified by RT-ROL, but is 39 shifted by a few nucleotides with respect to the extendible region+ It has been previously shown (Lima & Crooke, 1997) that the footprint generated by RNase H cleavage of heteroduplex RNA is slightly 39 shifted relative to the heteroduplex site+ On the other hand, extendible sites identified by RT-ROL, shown in Figure 5A, indicate only the positions of the 39 ends of the random oligonucleotides+ Therefore, the RNA region that can form a heteroduplex is expected FIGURE 5. Mapping of extendible sites in the ha-ras RNA stem-loop fragment+ A: Proposed secondary structure of the 47-nt stem-loop region corresponding to nt 18-64 of ha-ras mRNA+ Positions 18-25 of the loop that are favorable for extension with the random libraries are shown in bold face+ B: Intensities of the RT-ROL products obtained for the extendible site 18-25 of the ha-ras RNA stem-loop+ C: Association constants (K a , M Ϫ1 ) measured for 10-mer oligonucleotides complementary to the ha-ras RNA using the gel-shift assay+ The probes are named according to the positions of the targeted regions in the ha-ras RNA fragment+ to be 6-8 nt longer at its 39 end than the extendible region+ Taking both factors into account, we can conclude that the extendible sites in the ha-ras RNA stemloop determined by RT-ROL are in accordance with the accessible sites identified by RNase H assay + To demonstrate directly that extendible sites identify regions favorable for heteroduplex formation, we measured the association constants, K a , between 26 decanucleotides and the ha-ras RNA stem-loop using a gel-shift assay (see Materials and Methods)+ The K a values (Fig+ 5C) agree with the extendible sites shown in Figure 5A and with the results reported by Lima et al+ (1992)+ The decanucleotides that have 39 ends located within the extendible site 18-25 bind with the ha-ras RNA fragment much more strongly than do the rest of the oligonucleotides tested, with the exception of decamers 6-15 and 34-43+ The extendibility of the RNA region to which decanucleotide 6-15 binds cannot be determined by RT-ROL, because it overlaps with the RNA-specific PCR primer+ Although oligonucleotide 34-43 does bind to a nonextendible region in the stem duplex, this can be explained by the difference in lengths between decamer 34-43 and the optimal length (6-8 nt) for RT-ROL+ The greater length of the decamer could allow it to compete successfully with stem duplex formation+ Rabbit b-globin RNA The accessibility of rabbit b-globin RNA was previously analyzed using arrays of oligonucleotides up to 17 bases long complementary to bases 1-122 of the RNA sequence (Milner et al+, 1997)+ RNA binding was detected for 14-to 17-mer oligonucleotides that had the 39 ends located in regions 38-73 and 93-116+ No hybrid formation was detected in any other regions on the molecule+ Maximal hybridization with 17-mer libraries was observed for oligonucleotides that had the 39 ends located between nt 44-46+…”