Human RNase H1 Uses One Tryptophan and Two Lysines to Position the Enzyme at the 3′-DNA/5′-RNA Terminus of the Heteroduplex Substrate (2003)

In a previous study, we showed that the RNA-binding domain of human RNase H1 is responsible for the positional preference for cleavage exhibited by the enzyme (Wu, H., Lima, W. F., and Crooke, S. T. (2001) J. Biol. Chem. 276, 23547–23553). Here, we identify the substituents on the heteroduplex substrate and the amino acid residues within the RNA-binding domain of human RNase H1 involved in positioning of the enzyme. The human RNase H1 cleavage patterns observed for heteroduplexes with various 3′-DNA/5′-RNA and 5′-DNA/3′-RNA termini indicate that the 5′-most cleavage site on the oligoribonucleotide is positioned 7 bp from the first 3′-DNA/5′-RNA base pair. The presence or absence of phosphate or hydroxyl groups at either the 3′-DNA or 5′-RNA terminus had no effect on the human RNase H1 cleavage pattern. Substitution of the 3′-deoxynucleotide with a ribonucleotide, 2′-methoxyethyl nucleotide, or mismatched deoxyribonucleotide resulted in the ablation of the 5′-most cleavage site on the oligoribonucleotide. Mutants in which Trp43 and Lys59-Lys60 of the RNA-binding domain were substituted with alanine showed a loss of the positional preference for cleavage. Comparison of the kcat, Km, and Kd for the alanine-substituted mutants with those for human RNase H1 suggests that Lys59 and Lys60 are involved in binding to the heteroduplex and that Trp43 is responsible for properly positioning the enzyme on the substrate for catalysis. These data suggest that Trp43, Lys59, and Lys60 constitute an extended nucleic binding surface for the RNA-binding domain of human RNase H1, with the entire interaction taking place at the 3′-DNA/5′-RNA pole of the heteroduplex. These results offer further insights into the interaction between human RNase H1 and the heteroduplex substrate as well as approaches to enhance the design of effective antisense oligonucleotides. In a previous study, we showed that the RNA-binding domain of human RNase H1 is responsible for the positional preference for cleavage exhibited by the enzyme (Wu, H., Lima, W. F., and Crooke, S. T. (2001) J. Biol. Chem. 276, 23547–23553). Here, we identify the substituents on the heteroduplex substrate and the amino acid residues within the RNA-binding domain of human RNase H1 involved in positioning of the enzyme. The human RNase H1 cleavage patterns observed for heteroduplexes with various 3′-DNA/5′-RNA and 5′-DNA/3′-RNA termini indicate that the 5′-most cleavage site on the oligoribonucleotide is positioned 7 bp from the first 3′-DNA/5′-RNA base pair. The presence or absence of phosphate or hydroxyl groups at either the 3′-DNA or 5′-RNA terminus had no effect on the human RNase H1 cleavage pattern. Substitution of the 3′-deoxynucleotide with a ribonucleotide, 2′-methoxyethyl nucleotide, or mismatched deoxyribonucleotide resulted in the ablation of the 5′-most cleavage site on the oligoribonucleotide. Mutants in which Trp43 and Lys59-Lys60 of the RNA-binding domain were substituted with alanine showed a loss of the positional preference for cleavage. Comparison of the kcat, Km, and Kd for the alanine-substituted mutants with those for human RNase H1 suggests that Lys59 and Lys60 are involved in binding to the heteroduplex and that Trp43 is responsible for properly positioning the enzyme on the substrate for catalysis. These data suggest that Trp43, Lys59, and Lys60 constitute an extended nucleic binding surface for the RNA-binding domain of human RNase H1, with the entire interaction taking place at the 3′-DNA/5′-RNA pole of the heteroduplex. These results offer further insights into the interaction between human RNase H1 and the heteroduplex substrate as well as approaches to enhance the design of effective antisense oligonucleotides. RNase H hydrolyzes RNA in RNA/DNA hybrids (1.Stein H. Hausen P. Science. 1969; 166: 393-395Crossref PubMed Scopus (183) Google Scholar). RNase H activity appears to be ubiquitous in eukaryotes and bacteria (2.Itaya M. Kondo K. Nucleic Acids Res. 1991; 19: 4443-4449Crossref PubMed Scopus (63) Google Scholar, 3.Itaya M. McKelvin D. Chatterjie S.K. Crouch R.J. Mol. Gen. Genet. 1991; 227: 438-445Crossref PubMed Scopus (55) Google Scholar, 4.Kanaya S. Itaya M. J. Biol. Chem. 1992; 267: 10184-10192Abstract Full Text PDF PubMed Google Scholar, 5.Busen W. J. Biol. 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Antisense Nucleic Acid Drug Dev. 1998; 8: 53-61Crossref PubMed Scopus (55) Google Scholar, 10.Frank P. Braunshofer-Reiter C. Wintersberger U. Grimm R. Busen W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12872-12877Crossref PubMed Scopus (68) Google Scholar, 11.Cerritelli S.M. Crouch R.J. Genomics. 1998; 53: 307-311Crossref Scopus (48) Google Scholar). RNase H1 is a 286-amino acid protein and is expressed ubiquitously in human cells and tissues (9.Wu H. Lima W.F. Crooke S.T. Antisense Nucleic Acid Drug Dev. 1998; 8: 53-61Crossref PubMed Scopus (55) Google Scholar). The amino acid sequence of human RNase H1 displays strong homology to RNase H1 from yeast, chicken, Escherichia coli, and mouse (9.Wu H. Lima W.F. Crooke S.T. Antisense Nucleic Acid Drug Dev. 1998; 8: 53-61Crossref PubMed Scopus (55) Google Scholar). The human RNase H2 enzyme is a 299-amino acid protein with a calculated mass of 33.4 kDa and has also been shown to be ubiquitously expressed in human cells and tissues (10.Frank P. Braunshofer-Reiter C. Wintersberger U. Grimm R. Busen W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12872-12877Crossref PubMed Scopus (68) Google Scholar). 1H. Wu, unpublished data. Human RNase H2 shares strong amino acid sequence homology with RNase H2 from Caenorhabditis elegans, yeast, and E. coli (10.Frank P. Braunshofer-Reiter C. Wintersberger U. Grimm R. Busen W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12872-12877Crossref PubMed Scopus (68) Google Scholar). The structure of human RNase H1 was shown to consist of a 73-amino acid region homologous to the RNA-binding domain of yeast RNase H1 at the amino terminus of the protein and separated from the conserved catalytic domain by a 62-amino acid spacer region (12.Wu H. Lima W.F. Crooke S.T. J. Biol. Chem. 2001; 276: 23547-23553Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 13.Cerritelli S.M. Crouch R.J. RNA (N. Y.). 1995; 1: 246-259PubMed Google Scholar, 14.Evans S.P. Bycroft M. J. Mol. Biol. 1999; 291: 661-669Crossref PubMed Scopus (30) Google Scholar). The catalytic domain is highly conserved in the amino acid sequences of other RNase H1 proteins and contains the key catalytic and substrate-binding residues required for activity (12.Wu H. Lima W.F. Crooke S.T. J. Biol. Chem. 2001; 276: 23547-23553Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 15.Kanaya S. Katsuda-Kakai C. Ikehara M. J. Biol. Chem. 1991; 266: 11621-11627Abstract Full Text PDF PubMed Google Scholar, 16.Nakamura H. Oda Y. Iwai S. Inoue H. Ohtsuka E. Kanaya S. Kimura S. Katsuda C. Katayanagi K. Morikawa K. Miyashiro H. Ikehara M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11535-11539Crossref PubMed Scopus (196) Google Scholar, 17.Katayanagi K. Miyagawa M. Matsushima M. Ishkiawa M. Kanaya S. Ikehara M. Matsuzaki T. Morikawa K. Nature. 1990; 347: 306-309Crossref PubMed Scopus (308) Google Scholar, 18.Yang W. Hendrickson W.A. Crouch R.J. Satow Y. Science. 1990; 249: 1398-1405Crossref PubMed Scopus (453) Google Scholar). Site-directed mutagenesis of human RNase H1 revealed that the spacer region is required for RNase H activity, whereas the RNA-binding domain is not required for RNase H activity, i.e. RNA-binding domain deletion mutants exhibit RNase H activities comparable to those of the wild-type enzyme (12.Wu H. Lima W.F. Crooke S.T. J. Biol. Chem. 2001; 276: 23547-23553Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The RNA-binding domain of human RNase H1 is conserved in other eukaryotic RNases H1 (Fig. 1A) (9.Wu H. Lima W.F. Crooke S.T. Antisense Nucleic Acid Drug Dev. 1998; 8: 53-61Crossref PubMed Scopus (55) Google Scholar, 14.Evans S.P. Bycroft M. J. Mol. Biol. 1999; 291: 661-669Crossref PubMed Scopus (30) Google Scholar). The highly conserved lysines at positions 59 and 60 of the human enzyme have been shown in a 46-amino acid peptide of Saccharomyces cerevisiae RNase H1 to be important for binding to the heteroduplex substrate (13.Cerritelli S.M. Crouch R.J. RNA (N. Y.). 1995; 1: 246-259PubMed Google Scholar). The NMR structure of the RNA-binding domain of S. cerevisiae RNase H1 consists of a triple-stranded antiparallel β-sheet sandwiched between two α-helices and shares strong structural similarities with the N-terminal domain of the ribosomal RNA-binding protein L9 (14.Evans S.P. Bycroft M. J. Mol. Biol. 1999; 291: 661-669Crossref PubMed Scopus (30) Google Scholar, 19.Hoffman D.W. Cameron C.S. Davies C. White S.W. Ramakrishnan V. J. Mol. Biol. 1996; 264: 1058-1071Crossref PubMed Scopus (74) Google Scholar). The conserved lysine residues are located within the third β-strand. In addition, a highly conserved at was shown to amino acid at was also observed in the L9 protein and has been shown to be important for binding to ribosomal RNA D.W. Cameron C.S. Davies C. White S.W. Ramakrishnan V. J. Mol. Biol. 1996; 264: 1058-1071Crossref PubMed Scopus (74) Google Scholar). The of the RNA-binding domain of human RNase H1 is not Although the RNA-binding domain was shown not to be required for RNase H activity, region is responsible for the binding of the human enzyme for the heteroduplex substrate as well as the strong positional preference for cleavage exhibited by the i.e. human RNase H1 the heteroduplex substrate between 7 and from the 3′-DNA/5′-RNA terminus (12.Wu H. Lima W.F. Crooke S.T. J. Biol. Chem. 2001; 276: 23547-23553Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The positional preference for cleavage exhibited by the enzyme is with the for RNase H1, the of RNA S.M. C. A. Crouch R.J. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar, W. Hausen P. J. PubMed Scopus Google Scholar). The of the RNA (e.g. well with the observed cleavage specificity exhibited by human RNase H1 and suggests for the RNA-binding domain J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the RNA-binding domain the terminus of the heteroduplex substrate and positions the enzyme to further insights into the of the RNA-binding domain with to RNase H1 activity, the for the and the design of antisense oligonucleotides. In we heteroduplex with various 3′-DNA/5′-RNA and 5′-DNA/3′-RNA termini as well as 3′-DNA/5′-RNA termini to identify the substituents on the substrate important for positioning of human RNase In addition, mutants of the highly conserved Trp43, Lys59, and Lys60 residues of the RNA-binding domain of human RNase H1 were substituted with alanine to the of amino with to the positional preference for cleavage exhibited by the enzyme. of Human RNase H1 mutagenesis of human RNase H1 was a from H. U. 1990; PubMed Scopus Google Scholar). two were a of and two W.F. H. Crooke S.T. 2001; PubMed Scopus Google Scholar). For the RNase the for was and the was The for the RNase were and For the RNase the from the RNase was as a for a of with the for the RNase of were as W.F. H. Crooke S.T. 2001; PubMed Scopus Google Scholar). The was and cloned into the in proteins W.F. H. Crooke S.T. 2001; PubMed Scopus Google Scholar). The of the was by of Human RNase H1 were into E. coli The bacteria were in at and at The cells were with at for The cells were in and for at The proteins were for with of acid of The acid was into an protein and the RNase H1 proteins were with and and and The was further by and and RNase H1 were and The protein was by in and in and were in and by R. J.A. K. in Scholar). protein of were on a and J. Chem. 1998; Scopus Google Scholar). The were by The were on a by The were by from with of The were with either the 3′-deoxynucleotide or of RNA substrate was with of of of and The was at for The oligoribonucleotide was by on a J. T. Laboratory Cold Spring Harbor Cold Spring Scholar). The activity of the is of the heteroduplex substrate was in oligoribonucleotide from to and and were at for and to and 60 of RNase and at a of were were for at and was human RNase H1 proteins were with and for at The heteroduplex substrate was with of enzyme at of the cleavage was at from to and by of and The were at for and on a and the substrate and were on a The of the was as a function of The cleavage was from the of RNA of the for the of the which in of the and data from at The cleavage were as a function of the substrate and the data were to the the The to the heteroduplex substrate at and RNase to the of the were and as for the with the that RNA and human RNase H1 proteins in from to were The RNA for site to the of for site by of were by W.F. Crooke S.T. Biochemistry. PubMed Scopus Google Scholar). Here, the cleavage was for the heteroduplex substrate at a of in the presence and absence of a substrate The heteroduplex substrate was as in a of and with oligoribonucleotide and in from to The substrate was in of and The of the substrate was in of the heteroduplex substrate and from to were at for and to RNase and were to the as were at for and the substrate was to the heteroduplex was at a of and the was with of human RNase H1 The were and as for The human RNase H1 cleavage for the heteroduplexes a oligoribonucleotide and in from to are shown in These heteroduplexes RNA at either the terminus (Fig. or terminus (Fig. with previous H. Lima Crooke S.T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google the human RNase H1 were observed between 7 and from the terminus of the oligoribonucleotide (Fig. the of the heteroduplex by the terminus of the and the of the had no effect on the either the positions or of the (Fig. In the of the heteroduplex from the terminus of the the positions and of the (Fig. For example, a at the terminus of the resulted in the ablation of the cleavage on the oligoribonucleotide and a in the cleavage pattern. The of from the resulted in similar in the cleavage pattern. a a of 7 bp was observed between the terminus and the cleavage. in which the 3′-DNA/5′-RNA termini either a phosphate or hydroxyl on either the or oligoribonucleotide exhibited similar cleavage patterns (Fig. Substitution of the of the with either a or resulted in the ablation of the cleavage on the oligoribonucleotide (Fig. at the 3′-DNA/5′-RNA terminus also resulted in the loss of the cleavage on the oligoribonucleotide (Fig. substrate These data suggest that the cleavage by human RNase H1 is by the first base on the 3′-DNA/5′-RNA terminus of the heteroduplex. data that the enzyme a base as from an or base and that phosphate hydroxyl groups at the 3′-DNA/5′-RNA terminus are to the enzyme. identify the amino responsible for the strong positional preference for cleavage exhibited by human RNase H1, mutants were in which alanine was substituted for the conserved lysine residues at positions 59 and 60 the conserved at and all conserved amino (Fig. The cleavage patterns for the wild-type enzyme as well as the RNase H1 mutants were by the RNA for on the heteroduplex. that the observed cleavage was the of the was with the enzyme in of the substrate The positions of the cleavage for wild-type human RNase H1 were with the positions of the cleavage observed for the wild-type enzyme (Fig. as be the positional preference for cleavage was (e.g. the of RNase H1 within from the terminus of the human RNase H1 mutants exhibited cleavage patterns the wild-type human enzyme (Fig. The RNase (Fig. exhibited the cleavage with cleavage from to from the terminus of the oligoribonucleotide. In the RNase not appear to exhibit a positional preference for cleavage (e.g. the cleavage at and from the terminus of the oligoribonucleotide were comparable to the cleavage at the from the terminus of the is comparable to the of a human RNase H1 in which the RNA-binding domain is (12.Wu H. Lima W.F. Crooke S.T. J. Biol. Chem. 2001; 276: 23547-23553Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The for wild-type and human RNase H1 are shown in the RNase and RNase mutants exhibited the for human RNase in the for the heteroduplex were observed for the RNase and RNase mutants with the wild-type enzyme. in the catalytic was observed for in an in the with the wild-type enzyme the RNase exhibited a and with human RNase H1 the RNase to to the heteroduplex with an comparable to that of the wild-type enzyme cleavage for wild-type and human RNase H1 H1 H1 H1 in a Human RNase H1 has been shown to exhibit a strong positional preference for cleavage in with E. coli RNase H1 H. Lima Crooke S.T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The human enzyme the heteroduplex substrate from the whereas E. coli RNase H1 a cleavage pattern. The RNA-binding domain of human RNase H1 was shown to be responsible for the positional preference for cleavage exhibited by the human as deletion of domain resulted in an E. coli RNase H1 cleavage (12.Wu H. Lima W.F. Crooke S.T. J. Biol. Chem. 2001; 276: 23547-23553Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). These data also suggest on the positional preference for cleavage exhibited by the human the RNA-binding domain with the 3′-DNA/5′-RNA terminus of the heteroduplex. with we have that human RNase H1 exhibited a similar positional preference for cleavage for a of heteroduplex sequences (Fig. The positions of the 3′-DNA/5′-RNA and 5′-DNA/3′-RNA termini were on a oligoribonucleotide by the or of the The heteroduplexes in from to and either or 5′-RNA In addition, a was to a heteroduplex with a 3′-deoxynucleotide (Fig. The human RNase H1 cleavage patterns observed for heteroduplexes indicate that the cleavage on the oligoribonucleotide is positioned 7 bp from the first 3′-DNA/5′-RNA base pair. The of the 5′-DNA/3′-RNA terminus to have no on the positional preference for as the from the 3′-DNA/5′-RNA terminus to the cleavage was The presence of 5′-RNA had no effect on the of the cleavage. In a 3′-DNA to in an the for site was to the observed for other cleavage within heteroduplex. a positional preference for cleavage was observed (Fig. The were with the enzyme in of the heteroduplex and a interaction between the enzyme and The that the positional preference was suggests a interaction between human RNase H1 and the the of the RNase H1 proteins to the heteroduplex in a as to the substrate from the The of the with either a or 2′-methoxyethyl resulted in the ablation of the cleavage (Fig. the and 2′-methoxyethyl in a which an to from the observed for RNA/DNA heteroduplexes R.T. K. M. Nucleic Acids Res. 2001; 29: PubMed Scopus Google Scholar). The of the RNA/DNA heteroduplex an in a and a with the R.T. K. M. Nucleic Acids Res. 2001; 29: PubMed Scopus Google Scholar). the loss of the cleavage on the oligoribonucleotide from the of the with either a or 2′-methoxyethyl be to a loss of enzyme binding at the 3′-DNA/5′-RNA terminus by the in the These are further by the heteroduplex a at the 3′-DNA/5′-RNA which resulted in the loss of the cleavage (Fig. substrate the 2′-methoxyethyl and groups with the as are to be positioned within the of the a region also to be the binding site for the enzyme K. M. Morikawa K. Genet. 1993; PubMed Scopus Google Scholar). data suggest that the positioning of the human RNase H1 cleavage on the oligoribonucleotide is the of the enzyme binding to a base within the 3′-DNA/5′-RNA pole of the heteroduplex. The observed positional preference for cleavage exhibited by human RNase H1 well with the for enzyme. that the homologous mouse RNase H1 enzyme is required for and a in S.M. C. A. Crouch R.J. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar). The mouse enzyme is to in the of RNA These RNA of a region at the terminus and of in the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). on the positional preference for cleavage exhibited by human RNase H1, cleavage of the structure at or the RNA/DNA the RNA human RNase H1 cleavage of the RNA/DNA heteroduplex produce the observed for The RNA-binding domain of human RNase H1 is highly conserved in eukaryotic RNases H1 (Fig. 1A) (9.Wu H. Lima W.F. Crooke S.T. Antisense Nucleic Acid Drug Dev. 1998; 8: 53-61Crossref PubMed Scopus (55) Google Scholar, 14.Evans S.P. Bycroft M. J. Mol. Biol. 1999; 291: 661-669Crossref PubMed Scopus (30) Google Scholar). domain contains highly conserved amino acid residues Lys59, and which are to have a (Fig. Mutants were in which Trp43, and all conserved amino acid residues were substituted with alanine RNase and RNase (Fig. The cleavage patterns for the mutants an of the positional preference for cleavage exhibited by the wild-type enzyme (Fig. The RNase resulted in a cleavage comparable to that observed for E. coli RNase H1 and the RNA-binding domain deletion of human RNase H1 (Fig. (12.Wu H. Lima W.F. Crooke S.T. J. Biol. Chem. 2001; 276: 23547-23553Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). These results indicate that Lys59, and Trp43 the key amino acid residues within the RNA-binding domain of human RNase H1 involved in the 3′-DNA/5′-RNA terminus of the heteroduplex. Lys59 and Lys60 of human RNase H1 appear to to heteroduplex as the RNase and RNase mutants exhibited a for the heteroduplex with the human enzyme In addition, a in the was observed for mutants with human RNase The in the for the RNase and RNase mutants is with the in the for the RNA-binding domain deletion of human RNase H1 (12.Wu H. Lima W.F. Crooke S.T. J. Biol. Chem. 2001; 276: 23547-23553Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The in the Kd for the RNA-binding domain deletion of human RNase H1 the in the Kd for the RNase and RNase mutants suggests that amino acid residues within the RNA-binding domain of the human enzyme to the binding interaction (12.Wu H. Lima W.F. Crooke S.T. J. Biol. Chem. 2001; 276: 23547-23553Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). the of the lysine residues appears to be limited to substrate as a loss of catalytic activity was observed for the RNase and RNase mutants with the wild-type enzyme These are with the of the lysine residues of yeast RNase H1, which have been shown to be involved in binding to the heteroduplex (13.Cerritelli S.M. Crouch R.J. RNA (N. Y.). 1995; 1: 246-259PubMed Google Scholar). The NMR structure for the 46-amino acid peptide of the RNA-binding domain of S. cerevisiae RNase H1 the conserved lysine residues and positioned within the third of the triple-stranded β-sheet (14.Evans S.P. Bycroft M. J. Mol. Biol. 1999; 291: 661-669Crossref PubMed Scopus (30) Google Scholar). The groups of and were shown to in an on the β-strand. the the is and well with the of the of the a region to be the binding site for the enzyme K. M. Morikawa K. Genet. 1993; PubMed Scopus Google Scholar). The the is at the of the within of the conserved lysine the lysine residues with the of the heteroduplex within the of the the for the of the groups on the conserved the binding are with the lysines with phosphate groups of the base of base or within the The within the RNA-binding domain of human RNase H1 appears to be important for properly positioning the enzyme on the heteroduplex for catalysis. The RNase was with human RNase the and for the wild-type enzyme were those for the RNase The RNase and human RNase H1 exhibited comparable Kd for the that the in the activity of the RNase is not to a in the binding for the substrate the in the and for the RNase is of binding between the enzyme and substrate and suggests Trp43, the enzyme in that are not to catalysis. The conserved of S. cerevisiae RNase H1 a structure in by the of the of the is (14.Evans S.P. Bycroft M. J. Mol. Biol. 1999; 291: 661-669Crossref PubMed Scopus (30) Google Scholar). RNA-binding proteins amino to with the of the RNA and the of the of S. cerevisiae RNase H1 suggest a similar for J. S.K. Full Text Full Text PDF PubMed Scopus Google Scholar, K. Nature. PubMed Scopus Google Scholar). Trp43 of human RNase H1 either with the of the heteroduplex or with the base (e.g. the of the the of the heteroduplex the of the within the conserved Trp43, Lys59, and Lys60 residues constitute an extended nucleic binding surface for the RNA-binding domain of human RNase H1, with the entire interaction taking place at the 3′-DNA/5′-RNA pole of the heteroduplex. In base be important for of Trp43, and the phosphate the be for the interaction with Lys59 and with the data (e.g. and 2′-methoxyethyl as well as mismatched base binding of the RNA-binding domain to the substrate at those positions (Fig. In addition, the of the all the important that binding are between the conserved lysines and the heteroduplex. Here, Trp43 be to the positioning of the lysines that binding is of Trp43 as have effect on the binding of the enzyme for the in binding a in the site of binding in a in appears to place at the 3′-DNA/5′-RNA pole of the heteroduplex and from the of cleavage (Fig. in the site of binding the cleavage site (Fig. in the positioning required at the cleavage site (Fig. The of positioning on nucleic acid is a Human RNase H1 appears to identify the first 3′-DNA/5′-RNA base to be by positioning the amino involved in binding (e.g. Trp43, Lys59, and and from the catalytic the observed for the spacer region (12.Wu H. Lima W.F. Crooke S.T. J. Biol. Chem. 2001; 276: 23547-23553Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). the enzyme is at the site and the is the catalytic be positioned to the be a that is by and other be and are Human RNase H1 has been shown to a in the of by antisense antisense are in and are to function in and in the of have Antisense (e.g. 2′-methoxyethyl or for binding for the RNA and The that the RNA-binding domain of human RNase H1 the 3′-DNA/5′-RNA terminus of the heteroduplex and positions the catalytic site from the terminus suggests a of design that enhance the of antisense oligonucleotides. For example, the of the deoxyribonucleotide region of the antisense be in the of In addition, at the of the antisense is to have or no effect on human RNase H1 activity, whereas within the in the ablation of cleavage data suggest that the of substituents to the antisense be at the