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Design of Novel Relaxase Substrates Based on Rolling Circle Replicases for Bioconjugation to DNA Nanostructures.

Sagredo S, de la Cruz F, Moncalián G - PLoS ONE (2016)

Bottom Line: Both protein families show structural similarity but limited amino acid identity.Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins.The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular e Instituto de Biomedicina y Biotecnología de Cantabria, Universidad de Cantabria-Consejo Superior de Investigaciones Científicas-SODERCAN, C/ Albert Einstein 22, 39011, Santander, Spain.

ABSTRACT
During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence (nic site). Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOBF, MOBQ and MOBP families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.

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Related in: MedlinePlus

Interaction of TrwCR with Reverse substrates.(A) Reverse substrates were designed by swapping the 5’ region of the nic site to the 3’ end. This designed DNA substrate possesses the complete inverted repeat (D-P) at the 3’ end of the nic site (N). Either the U or the S lengths were tuned to allow the correct location of the hairpin within the relaxase binding domain. (B) Scheme depicting the cleavage reaction of the reverse substrate. Relaxase binding to the reverse substrate allows both the hairpin and the single strand U-turn localize at the DNA binding cleft. This way the cleavage reaction forms a covalent complex of the relaxase with the region downstream of the nic site (blue arrowhead). Now the 5´side of nic do not contain the IR avoiding the re-ligation reaction. Color code is the same than Fig 1. (C) 12% SDS-PAGE of reverse oligonucleotides, when incubated with TrwCR. 6 μM TrwCR was incubated with 15 μM of different reverse oligonucleotides. Lane 1, no oligonucleotide; Lanes 2, R388wt oligonucleotide W(25+18). Lanes 3 and 4, Reverse substrates R(8+27) and R(8+24), both with U = 8 nt and S = 11nt or S = 8 nt respectively. Lane 5, R(7+27) U = 7; Lane 6, R(4+27) U = 4; Lane 7, R(1+27) U = 1 and Lane 8, R(0+27) U = 0. Lane 9, R(8+14), U = 8 P = 0. Lane 10, Molecular weight marker. Graph quantifying the percentages of covalent complexes is shown below the SDS-PAGE gel. Data show mean±s.d. of three independent experiments. Two asterisks indicate P-value<0.05 by two-sided student’s t-text.
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pone.0152666.g004: Interaction of TrwCR with Reverse substrates.(A) Reverse substrates were designed by swapping the 5’ region of the nic site to the 3’ end. This designed DNA substrate possesses the complete inverted repeat (D-P) at the 3’ end of the nic site (N). Either the U or the S lengths were tuned to allow the correct location of the hairpin within the relaxase binding domain. (B) Scheme depicting the cleavage reaction of the reverse substrate. Relaxase binding to the reverse substrate allows both the hairpin and the single strand U-turn localize at the DNA binding cleft. This way the cleavage reaction forms a covalent complex of the relaxase with the region downstream of the nic site (blue arrowhead). Now the 5´side of nic do not contain the IR avoiding the re-ligation reaction. Color code is the same than Fig 1. (C) 12% SDS-PAGE of reverse oligonucleotides, when incubated with TrwCR. 6 μM TrwCR was incubated with 15 μM of different reverse oligonucleotides. Lane 1, no oligonucleotide; Lanes 2, R388wt oligonucleotide W(25+18). Lanes 3 and 4, Reverse substrates R(8+27) and R(8+24), both with U = 8 nt and S = 11nt or S = 8 nt respectively. Lane 5, R(7+27) U = 7; Lane 6, R(4+27) U = 4; Lane 7, R(1+27) U = 1 and Lane 8, R(0+27) U = 0. Lane 9, R(8+14), U = 8 P = 0. Lane 10, Molecular weight marker. Graph quantifying the percentages of covalent complexes is shown below the SDS-PAGE gel. Data show mean±s.d. of three independent experiments. Two asterisks indicate P-value<0.05 by two-sided student’s t-text.

Mentions: In TrwCR wt substrate W(25+8), the stem loop is non-covalently bound by the relaxase after cleavage, while the 8-mer downstream nic is covalently attached to the catalytic tyrosine. There is a cleavage-ligation equilibrium with wt oligonucleotides, because the 25-mer oligonucleotide remains in the protein DNA binding domain after cleavage and, therefore, the 8-mer can be easily religated (Fig 1C). We thought that, by linking the 3´ end of nic (U-N-S) to the 5´end of the hairpin, the 5´side of nic (the single strand U sequence upstream the nic site) would lose binding affinity (Fig 4A). As a consequence, the resulting 5´ssDNA could be released from the relaxase, avoiding religation and displacing the reaction equilibrium towards the formation of covalent complexes (Fig 4B). Under this rationale, novel substrates were designed by shuffling the wt sequence in the order U-N-S-D-P. These synthetic oligonucleotides were called “reverse substrates” because the hairpin is located downstream from the nic site.


Design of Novel Relaxase Substrates Based on Rolling Circle Replicases for Bioconjugation to DNA Nanostructures.

Sagredo S, de la Cruz F, Moncalián G - PLoS ONE (2016)

Interaction of TrwCR with Reverse substrates.(A) Reverse substrates were designed by swapping the 5’ region of the nic site to the 3’ end. This designed DNA substrate possesses the complete inverted repeat (D-P) at the 3’ end of the nic site (N). Either the U or the S lengths were tuned to allow the correct location of the hairpin within the relaxase binding domain. (B) Scheme depicting the cleavage reaction of the reverse substrate. Relaxase binding to the reverse substrate allows both the hairpin and the single strand U-turn localize at the DNA binding cleft. This way the cleavage reaction forms a covalent complex of the relaxase with the region downstream of the nic site (blue arrowhead). Now the 5´side of nic do not contain the IR avoiding the re-ligation reaction. Color code is the same than Fig 1. (C) 12% SDS-PAGE of reverse oligonucleotides, when incubated with TrwCR. 6 μM TrwCR was incubated with 15 μM of different reverse oligonucleotides. Lane 1, no oligonucleotide; Lanes 2, R388wt oligonucleotide W(25+18). Lanes 3 and 4, Reverse substrates R(8+27) and R(8+24), both with U = 8 nt and S = 11nt or S = 8 nt respectively. Lane 5, R(7+27) U = 7; Lane 6, R(4+27) U = 4; Lane 7, R(1+27) U = 1 and Lane 8, R(0+27) U = 0. Lane 9, R(8+14), U = 8 P = 0. Lane 10, Molecular weight marker. Graph quantifying the percentages of covalent complexes is shown below the SDS-PAGE gel. Data show mean±s.d. of three independent experiments. Two asterisks indicate P-value<0.05 by two-sided student’s t-text.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4814116&req=5

pone.0152666.g004: Interaction of TrwCR with Reverse substrates.(A) Reverse substrates were designed by swapping the 5’ region of the nic site to the 3’ end. This designed DNA substrate possesses the complete inverted repeat (D-P) at the 3’ end of the nic site (N). Either the U or the S lengths were tuned to allow the correct location of the hairpin within the relaxase binding domain. (B) Scheme depicting the cleavage reaction of the reverse substrate. Relaxase binding to the reverse substrate allows both the hairpin and the single strand U-turn localize at the DNA binding cleft. This way the cleavage reaction forms a covalent complex of the relaxase with the region downstream of the nic site (blue arrowhead). Now the 5´side of nic do not contain the IR avoiding the re-ligation reaction. Color code is the same than Fig 1. (C) 12% SDS-PAGE of reverse oligonucleotides, when incubated with TrwCR. 6 μM TrwCR was incubated with 15 μM of different reverse oligonucleotides. Lane 1, no oligonucleotide; Lanes 2, R388wt oligonucleotide W(25+18). Lanes 3 and 4, Reverse substrates R(8+27) and R(8+24), both with U = 8 nt and S = 11nt or S = 8 nt respectively. Lane 5, R(7+27) U = 7; Lane 6, R(4+27) U = 4; Lane 7, R(1+27) U = 1 and Lane 8, R(0+27) U = 0. Lane 9, R(8+14), U = 8 P = 0. Lane 10, Molecular weight marker. Graph quantifying the percentages of covalent complexes is shown below the SDS-PAGE gel. Data show mean±s.d. of three independent experiments. Two asterisks indicate P-value<0.05 by two-sided student’s t-text.
Mentions: In TrwCR wt substrate W(25+8), the stem loop is non-covalently bound by the relaxase after cleavage, while the 8-mer downstream nic is covalently attached to the catalytic tyrosine. There is a cleavage-ligation equilibrium with wt oligonucleotides, because the 25-mer oligonucleotide remains in the protein DNA binding domain after cleavage and, therefore, the 8-mer can be easily religated (Fig 1C). We thought that, by linking the 3´ end of nic (U-N-S) to the 5´end of the hairpin, the 5´side of nic (the single strand U sequence upstream the nic site) would lose binding affinity (Fig 4A). As a consequence, the resulting 5´ssDNA could be released from the relaxase, avoiding religation and displacing the reaction equilibrium towards the formation of covalent complexes (Fig 4B). Under this rationale, novel substrates were designed by shuffling the wt sequence in the order U-N-S-D-P. These synthetic oligonucleotides were called “reverse substrates” because the hairpin is located downstream from the nic site.

Bottom Line: Both protein families show structural similarity but limited amino acid identity.Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins.The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular e Instituto de Biomedicina y Biotecnología de Cantabria, Universidad de Cantabria-Consejo Superior de Investigaciones Científicas-SODERCAN, C/ Albert Einstein 22, 39011, Santander, Spain.

ABSTRACT
During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence (nic site). Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOBF, MOBQ and MOBP families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.

Show MeSH
Related in: MedlinePlus