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Sequence-specific cleavage of the RNA strand in DNA-RNA hybrids by the fusion of ribonuclease H with a zinc finger.

Sulej AA, Tuszynska I, Skowronek KJ, Nowotny M, Bujnicki JM - Nucleic Acids Res. (2012)

Bottom Line: The optimization of the fusion enzyme's specificity was guided by a structural model of the protein-substrate complex and involved a number of steps, including site-directed mutagenesis of the RNase moiety and optimization of the interdomain linker length.Methods for engineering zinc finger domains with new sequence specificities are readily available, making it feasible to acquire a library of RNases that recognize and cleave a variety of sequences, much like the commercially available assortment of restriction enzymes.Potentially, zinc finger-RNase HI fusions may, in addition to in vitro applications, be used in vivo for targeted RNA degradation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, Ks. Trojdena Street 4, 02-109 Warsaw, Poland.

ABSTRACT
Ribonucleases (RNases) are valuable tools applied in the analysis of RNA sequence, structure and function. Their substrate specificity is limited to recognition of single bases or distinct secondary structures in the substrate. Currently, there are no RNases available for purely sequence-dependent fragmentation of RNA. Here, we report the development of a new enzyme that cleaves the RNA strand in DNA-RNA hybrids 5 nt from a nonanucleotide recognition sequence. The enzyme was constructed by fusing two functionally independent domains, a RNase HI, that hydrolyzes RNA in DNA-RNA hybrids in processive and sequence-independent manner, and a zinc finger that recognizes a sequence in DNA-RNA hybrids. The optimization of the fusion enzyme's specificity was guided by a structural model of the protein-substrate complex and involved a number of steps, including site-directed mutagenesis of the RNase moiety and optimization of the interdomain linker length. Methods for engineering zinc finger domains with new sequence specificities are readily available, making it feasible to acquire a library of RNases that recognize and cleave a variety of sequences, much like the commercially available assortment of restriction enzymes. Potentially, zinc finger-RNase HI fusions may, in addition to in vitro applications, be used in vivo for targeted RNA degradation.

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Structural model of the RNase HI-ZfQQR hybrid enzyme variants (catAEA-ZfQQR, GQ and GGKKQ) in complex with the DNA–RNA hybrid, with the cleavage site positioned 5 nt away from the ZfQQR binding site. Protein and nucleic acid backbone is shown in the cartoon representation: the RNase HI catalytic domain is shown in blue (Mg2+ ions are shown as cyan spheres), the ZfQQR module is in green (Zn2+ ions are shown as lime spheres), the DNA strand is shown in dark gray (with the sequence recognized by ZfQQR in light gray), and the RNA strand is in dark yellow (the sequence recognized by ZfQQR in light yellow). A phosphorus atom participating in the scissile phosphodiester bond is shown as a yellow sphere. The interdomain linkers of the catAEA-ZfQQR, GGKKQ and GQ variants are shown in yellow, orange and red, respectively. Sequences of the interdomain linkers for each variant are shown above the model.
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gks885-F2: Structural model of the RNase HI-ZfQQR hybrid enzyme variants (catAEA-ZfQQR, GQ and GGKKQ) in complex with the DNA–RNA hybrid, with the cleavage site positioned 5 nt away from the ZfQQR binding site. Protein and nucleic acid backbone is shown in the cartoon representation: the RNase HI catalytic domain is shown in blue (Mg2+ ions are shown as cyan spheres), the ZfQQR module is in green (Zn2+ ions are shown as lime spheres), the DNA strand is shown in dark gray (with the sequence recognized by ZfQQR in light gray), and the RNA strand is in dark yellow (the sequence recognized by ZfQQR in light yellow). A phosphorus atom participating in the scissile phosphodiester bond is shown as a yellow sphere. The interdomain linkers of the catAEA-ZfQQR, GGKKQ and GQ variants are shown in yellow, orange and red, respectively. Sequences of the interdomain linkers for each variant are shown above the model.

Mentions: To investigate the arrangement of the catalytic and substrate-binding domains in the fusion enzyme and to guide further engineering of the protein, we built a structural model of the catAEA-ZfQQR fusion enzyme complexed with the substrate (see Materials and Methods section for details). The catalytic domain was positioned to cleave in the most preferred location 5 nt away from the ZfQQR binding site (see Cleavage site mapping section for more details). The model (Figure 2) revealed that the initially used interdomain linker is longer than necessary and suggested that its shortening may decrease the cleavage at sites further away from the binding site. Based on the model, we designed two modifications, in which the interdomain linker was shortened by 11 and 8 amino acids, respectively. The resulting protein variants had only two (GQ) or five (GGKKQ) residues between the structurally important residue Y193 in the C-terminus of the RNase HI domain, and the N-terminal residue (H) of the first zinc finger domain in the ZfQQR module.Figure 2.


Sequence-specific cleavage of the RNA strand in DNA-RNA hybrids by the fusion of ribonuclease H with a zinc finger.

Sulej AA, Tuszynska I, Skowronek KJ, Nowotny M, Bujnicki JM - Nucleic Acids Res. (2012)

Structural model of the RNase HI-ZfQQR hybrid enzyme variants (catAEA-ZfQQR, GQ and GGKKQ) in complex with the DNA–RNA hybrid, with the cleavage site positioned 5 nt away from the ZfQQR binding site. Protein and nucleic acid backbone is shown in the cartoon representation: the RNase HI catalytic domain is shown in blue (Mg2+ ions are shown as cyan spheres), the ZfQQR module is in green (Zn2+ ions are shown as lime spheres), the DNA strand is shown in dark gray (with the sequence recognized by ZfQQR in light gray), and the RNA strand is in dark yellow (the sequence recognized by ZfQQR in light yellow). A phosphorus atom participating in the scissile phosphodiester bond is shown as a yellow sphere. The interdomain linkers of the catAEA-ZfQQR, GGKKQ and GQ variants are shown in yellow, orange and red, respectively. Sequences of the interdomain linkers for each variant are shown above the model.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526281&req=5

gks885-F2: Structural model of the RNase HI-ZfQQR hybrid enzyme variants (catAEA-ZfQQR, GQ and GGKKQ) in complex with the DNA–RNA hybrid, with the cleavage site positioned 5 nt away from the ZfQQR binding site. Protein and nucleic acid backbone is shown in the cartoon representation: the RNase HI catalytic domain is shown in blue (Mg2+ ions are shown as cyan spheres), the ZfQQR module is in green (Zn2+ ions are shown as lime spheres), the DNA strand is shown in dark gray (with the sequence recognized by ZfQQR in light gray), and the RNA strand is in dark yellow (the sequence recognized by ZfQQR in light yellow). A phosphorus atom participating in the scissile phosphodiester bond is shown as a yellow sphere. The interdomain linkers of the catAEA-ZfQQR, GGKKQ and GQ variants are shown in yellow, orange and red, respectively. Sequences of the interdomain linkers for each variant are shown above the model.
Mentions: To investigate the arrangement of the catalytic and substrate-binding domains in the fusion enzyme and to guide further engineering of the protein, we built a structural model of the catAEA-ZfQQR fusion enzyme complexed with the substrate (see Materials and Methods section for details). The catalytic domain was positioned to cleave in the most preferred location 5 nt away from the ZfQQR binding site (see Cleavage site mapping section for more details). The model (Figure 2) revealed that the initially used interdomain linker is longer than necessary and suggested that its shortening may decrease the cleavage at sites further away from the binding site. Based on the model, we designed two modifications, in which the interdomain linker was shortened by 11 and 8 amino acids, respectively. The resulting protein variants had only two (GQ) or five (GGKKQ) residues between the structurally important residue Y193 in the C-terminus of the RNase HI domain, and the N-terminal residue (H) of the first zinc finger domain in the ZfQQR module.Figure 2.

Bottom Line: The optimization of the fusion enzyme's specificity was guided by a structural model of the protein-substrate complex and involved a number of steps, including site-directed mutagenesis of the RNase moiety and optimization of the interdomain linker length.Methods for engineering zinc finger domains with new sequence specificities are readily available, making it feasible to acquire a library of RNases that recognize and cleave a variety of sequences, much like the commercially available assortment of restriction enzymes.Potentially, zinc finger-RNase HI fusions may, in addition to in vitro applications, be used in vivo for targeted RNA degradation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, Ks. Trojdena Street 4, 02-109 Warsaw, Poland.

ABSTRACT
Ribonucleases (RNases) are valuable tools applied in the analysis of RNA sequence, structure and function. Their substrate specificity is limited to recognition of single bases or distinct secondary structures in the substrate. Currently, there are no RNases available for purely sequence-dependent fragmentation of RNA. Here, we report the development of a new enzyme that cleaves the RNA strand in DNA-RNA hybrids 5 nt from a nonanucleotide recognition sequence. The enzyme was constructed by fusing two functionally independent domains, a RNase HI, that hydrolyzes RNA in DNA-RNA hybrids in processive and sequence-independent manner, and a zinc finger that recognizes a sequence in DNA-RNA hybrids. The optimization of the fusion enzyme's specificity was guided by a structural model of the protein-substrate complex and involved a number of steps, including site-directed mutagenesis of the RNase moiety and optimization of the interdomain linker length. Methods for engineering zinc finger domains with new sequence specificities are readily available, making it feasible to acquire a library of RNases that recognize and cleave a variety of sequences, much like the commercially available assortment of restriction enzymes. Potentially, zinc finger-RNase HI fusions may, in addition to in vitro applications, be used in vivo for targeted RNA degradation.

Show MeSH
Related in: MedlinePlus