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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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Interaction of TrwCR with Rep-like substrates.(A) SDS-PAGE of oligonucleotides with R388wt or Rep-Like structures, when incubated with TrwCR. 6 μM TrwCR was incubated with 15 μM of different oligonucleotides. The reaction products were separated by electrophoresis in 12% SDS-PAGE gels. Lane 1, no oligonucleotide; Lanes 2 and 3, R388wt oligonucleotides W(25+18) and W(14+14), respectively; in subsequent lanes, TrwCR was incubated with Rep-like oligonucleotides. The S length of Rep-like substrates (in green), varies from two to eleven nucleotides. Lane 4, H(14+8), Lane 5, H(14+10), Lane 6, H(14+12); Lane 7, H(14+13), Lane 8, H(14+14); Lane 9, H(14+15); Lane 10, H(14+17). In the center chart, percentage of bound complexes were calculated in three separate experiments such as that shown in (A). (B) Increasing amounts of TrwCR were incubated with wt oligonucleotide W(25+8) (red shift, lanes 1 to 8) or Rep-like hairpin H(14+14) (green shift, lanes 9–16). Lanes 1 and 9, no protein added; 2 and 10, 42 nM of TrwCR; 3 and 11, 85 nM; 4 and 12, 210 nM; 5 and 13, 420 nM; 6 and 14,850 nM; 7 and 15, 4,2 μM, 8 and 16, 8,5 μM.
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pone.0152666.g002: Interaction of TrwCR with Rep-like substrates.(A) SDS-PAGE of oligonucleotides with R388wt or Rep-Like structures, when incubated with TrwCR. 6 μM TrwCR was incubated with 15 μM of different oligonucleotides. The reaction products were separated by electrophoresis in 12% SDS-PAGE gels. Lane 1, no oligonucleotide; Lanes 2 and 3, R388wt oligonucleotides W(25+18) and W(14+14), respectively; in subsequent lanes, TrwCR was incubated with Rep-like oligonucleotides. The S length of Rep-like substrates (in green), varies from two to eleven nucleotides. Lane 4, H(14+8), Lane 5, H(14+10), Lane 6, H(14+12); Lane 7, H(14+13), Lane 8, H(14+14); Lane 9, H(14+15); Lane 10, H(14+17). In the center chart, percentage of bound complexes were calculated in three separate experiments such as that shown in (A). (B) Increasing amounts of TrwCR were incubated with wt oligonucleotide W(25+8) (red shift, lanes 1 to 8) or Rep-like hairpin H(14+14) (green shift, lanes 9–16). Lanes 1 and 9, no protein added; 2 and 10, 42 nM of TrwCR; 3 and 11, 85 nM; 4 and 12, 210 nM; 5 and 13, 420 nM; 6 and 14,850 nM; 7 and 15, 4,2 μM, 8 and 16, 8,5 μM.

Mentions: The effect of the DNA substrate length and secondary structure on relaxase cleavage was investigated through in vitro nic-cleavage reactions. This reaction generates a protein-DNA covalent complex that can be quantified by its lower mobility using SDS-PAGE as described in Materials and Methods. TrwCR was able to cleave and remain covalently bound to oligonucleotides containing IR2-nic (W(25+18)) or P-nic (W(14+14)), (Fig 2A, Lanes 2 and 3). As previously described, the entire IR2 increases the binding affinity of TrwCR but the distal arm is not required for efficient cleavage (9). Interestingly, TrwCR could also cleave a Rep-like oligonucleotide containing a loop with only 2 nucleotides in the S region (H(14+8)) (Fig 2, lane 4). TrwCR cleavage activity on H(14+8) was similar to that on W oligonucleotides. Incubation of TrwCR with Rep-like hairpins containing longer loops, such as H(14+10), H(14+12) or H(14+13), resulted in a band with reduced mobility, as expected by the formation of a TrwCR covalent complex with a decamer (Lane 5), dodecamer (Lane 6) and tridecamer (Lane 7), respectively. Similar results were obtained with substrates H(14+14), H(14+15) or H(14+17) (Lanes 8, 9 and 10, respectively). Labeled Rep-like oligonucleotide H14+14 was also found to be effectively cleaved by TrwCR using TBE-urea gel electrophoresis analysis (S2 Fig).


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 Rep-like substrates.(A) SDS-PAGE of oligonucleotides with R388wt or Rep-Like structures, when incubated with TrwCR. 6 μM TrwCR was incubated with 15 μM of different oligonucleotides. The reaction products were separated by electrophoresis in 12% SDS-PAGE gels. Lane 1, no oligonucleotide; Lanes 2 and 3, R388wt oligonucleotides W(25+18) and W(14+14), respectively; in subsequent lanes, TrwCR was incubated with Rep-like oligonucleotides. The S length of Rep-like substrates (in green), varies from two to eleven nucleotides. Lane 4, H(14+8), Lane 5, H(14+10), Lane 6, H(14+12); Lane 7, H(14+13), Lane 8, H(14+14); Lane 9, H(14+15); Lane 10, H(14+17). In the center chart, percentage of bound complexes were calculated in three separate experiments such as that shown in (A). (B) Increasing amounts of TrwCR were incubated with wt oligonucleotide W(25+8) (red shift, lanes 1 to 8) or Rep-like hairpin H(14+14) (green shift, lanes 9–16). Lanes 1 and 9, no protein added; 2 and 10, 42 nM of TrwCR; 3 and 11, 85 nM; 4 and 12, 210 nM; 5 and 13, 420 nM; 6 and 14,850 nM; 7 and 15, 4,2 μM, 8 and 16, 8,5 μM.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4814116&req=5

pone.0152666.g002: Interaction of TrwCR with Rep-like substrates.(A) SDS-PAGE of oligonucleotides with R388wt or Rep-Like structures, when incubated with TrwCR. 6 μM TrwCR was incubated with 15 μM of different oligonucleotides. The reaction products were separated by electrophoresis in 12% SDS-PAGE gels. Lane 1, no oligonucleotide; Lanes 2 and 3, R388wt oligonucleotides W(25+18) and W(14+14), respectively; in subsequent lanes, TrwCR was incubated with Rep-like oligonucleotides. The S length of Rep-like substrates (in green), varies from two to eleven nucleotides. Lane 4, H(14+8), Lane 5, H(14+10), Lane 6, H(14+12); Lane 7, H(14+13), Lane 8, H(14+14); Lane 9, H(14+15); Lane 10, H(14+17). In the center chart, percentage of bound complexes were calculated in three separate experiments such as that shown in (A). (B) Increasing amounts of TrwCR were incubated with wt oligonucleotide W(25+8) (red shift, lanes 1 to 8) or Rep-like hairpin H(14+14) (green shift, lanes 9–16). Lanes 1 and 9, no protein added; 2 and 10, 42 nM of TrwCR; 3 and 11, 85 nM; 4 and 12, 210 nM; 5 and 13, 420 nM; 6 and 14,850 nM; 7 and 15, 4,2 μM, 8 and 16, 8,5 μM.
Mentions: The effect of the DNA substrate length and secondary structure on relaxase cleavage was investigated through in vitro nic-cleavage reactions. This reaction generates a protein-DNA covalent complex that can be quantified by its lower mobility using SDS-PAGE as described in Materials and Methods. TrwCR was able to cleave and remain covalently bound to oligonucleotides containing IR2-nic (W(25+18)) or P-nic (W(14+14)), (Fig 2A, Lanes 2 and 3). As previously described, the entire IR2 increases the binding affinity of TrwCR but the distal arm is not required for efficient cleavage (9). Interestingly, TrwCR could also cleave a Rep-like oligonucleotide containing a loop with only 2 nucleotides in the S region (H(14+8)) (Fig 2, lane 4). TrwCR cleavage activity on H(14+8) was similar to that on W oligonucleotides. Incubation of TrwCR with Rep-like hairpins containing longer loops, such as H(14+10), H(14+12) or H(14+13), resulted in a band with reduced mobility, as expected by the formation of a TrwCR covalent complex with a decamer (Lane 5), dodecamer (Lane 6) and tridecamer (Lane 7), respectively. Similar results were obtained with substrates H(14+14), H(14+15) or H(14+17) (Lanes 8, 9 and 10, respectively). Labeled Rep-like oligonucleotide H14+14 was also found to be effectively cleaved by TrwCR using TBE-urea gel electrophoresis analysis (S2 Fig).

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