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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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Novel designs for DNA substrates of model single-Y relaxases.(A) SDS-PAGE of MobAR_RSF1010 with its targets. 7 μM MobAR was incubated with 15 μM of different oligonucleotides. Lane 1, MobAR. Lanes 2 and 3, wt oligonucleotide WQ(30+7) and a substrate that lacks the upper-hairpin nucleotides of the IR WQ(23+7). Lanes 4, 5 and 6, Rep-like oligonucleotides HQ(16+16),HQ(16+19) and HQ(16+22), respectively. Lanes 7, 8 and 9 reverse substrates RQ(8+28), RQ(8+34) and RQ(8+40). Lane 10, molecular weight ladder. (B) SDS-PAGE of TraIR_RP4 with its targets. 1.5 μM TraIR was incubated with 15 μM of different oligonucleotides. Lane 1, TraIR; lane 2, wt substrate WP(15+6); lane 3, wt substrate WP(24+8); lane 4, reverse substrate RP(8+24) and lane 5, Rep-like substrate HP(14+14). Molecular weight ladder is shown on Lane 6. Bar graphs with the quantification of covalent complexes are shown below the SDS-PAGE gels. Data showed mean±s.d. of three independent experiments.
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pone.0152666.g003: Novel designs for DNA substrates of model single-Y relaxases.(A) SDS-PAGE of MobAR_RSF1010 with its targets. 7 μM MobAR was incubated with 15 μM of different oligonucleotides. Lane 1, MobAR. Lanes 2 and 3, wt oligonucleotide WQ(30+7) and a substrate that lacks the upper-hairpin nucleotides of the IR WQ(23+7). Lanes 4, 5 and 6, Rep-like oligonucleotides HQ(16+16),HQ(16+19) and HQ(16+22), respectively. Lanes 7, 8 and 9 reverse substrates RQ(8+28), RQ(8+34) and RQ(8+40). Lane 10, molecular weight ladder. (B) SDS-PAGE of TraIR_RP4 with its targets. 1.5 μM TraIR was incubated with 15 μM of different oligonucleotides. Lane 1, TraIR; lane 2, wt substrate WP(15+6); lane 3, wt substrate WP(24+8); lane 4, reverse substrate RP(8+24) and lane 5, Rep-like substrate HP(14+14). Molecular weight ladder is shown on Lane 6. Bar graphs with the quantification of covalent complexes are shown below the SDS-PAGE gels. Data showed mean±s.d. of three independent experiments.

Mentions: Relaxases contain either one (Y1) or two (Y2) catalytic tyrosines [1,12]. The ssDNA U-turn observed in Y2 relaxase domains TrwCR- and TraIR -DNA complex structures was also observed in the structure of Y1 relaxase NES [13–15]. However, relevant differences in U-turn formation have been described [12]. In order to check if Y1 relaxases could also catalyze cleavage of Rep-like substrates, we analyzed these substrates for the best known Y1-relaxases, MobA_RSF1010 and TraI_RP4 (S7 Fig). The MobA_RSF1010 binding site was identified as a 10 bp IR that forms a hairpin structure upstream from nic [16]. Mozingo et al. modeled the relaxase minMobA (amino acids 1 to 186, named MobAR in this study) complexed with a 33-mer oligonucleotide and compared this model with TrwCR bound to the 23-mer [16]. According to this comparison, only nucleotides at the base of the 10 bp-hairpin interact with MobAR. To study the influence of IR length, we designed a shorter wt substrate, containing a 6 bp IR WQ(23+7) (Fig 3A and Table 2). Analysis by SDS-PAGE of the covalent complexes obtained after incubation with the protein showed that either the long wt substrate WQ(30+7) (Fig 3A Lane 2) or the short wt substrate WQ(23+7) (Fig 3A Lane 3) generated 40% covalent complexes. These results confirm that just the nucleotides at the base of the stem make specific interactions with relaxase MobAR.


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)

Novel designs for DNA substrates of model single-Y relaxases.(A) SDS-PAGE of MobAR_RSF1010 with its targets. 7 μM MobAR was incubated with 15 μM of different oligonucleotides. Lane 1, MobAR. Lanes 2 and 3, wt oligonucleotide WQ(30+7) and a substrate that lacks the upper-hairpin nucleotides of the IR WQ(23+7). Lanes 4, 5 and 6, Rep-like oligonucleotides HQ(16+16),HQ(16+19) and HQ(16+22), respectively. Lanes 7, 8 and 9 reverse substrates RQ(8+28), RQ(8+34) and RQ(8+40). Lane 10, molecular weight ladder. (B) SDS-PAGE of TraIR_RP4 with its targets. 1.5 μM TraIR was incubated with 15 μM of different oligonucleotides. Lane 1, TraIR; lane 2, wt substrate WP(15+6); lane 3, wt substrate WP(24+8); lane 4, reverse substrate RP(8+24) and lane 5, Rep-like substrate HP(14+14). Molecular weight ladder is shown on Lane 6. Bar graphs with the quantification of covalent complexes are shown below the SDS-PAGE gels. Data showed mean±s.d. of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152666.g003: Novel designs for DNA substrates of model single-Y relaxases.(A) SDS-PAGE of MobAR_RSF1010 with its targets. 7 μM MobAR was incubated with 15 μM of different oligonucleotides. Lane 1, MobAR. Lanes 2 and 3, wt oligonucleotide WQ(30+7) and a substrate that lacks the upper-hairpin nucleotides of the IR WQ(23+7). Lanes 4, 5 and 6, Rep-like oligonucleotides HQ(16+16),HQ(16+19) and HQ(16+22), respectively. Lanes 7, 8 and 9 reverse substrates RQ(8+28), RQ(8+34) and RQ(8+40). Lane 10, molecular weight ladder. (B) SDS-PAGE of TraIR_RP4 with its targets. 1.5 μM TraIR was incubated with 15 μM of different oligonucleotides. Lane 1, TraIR; lane 2, wt substrate WP(15+6); lane 3, wt substrate WP(24+8); lane 4, reverse substrate RP(8+24) and lane 5, Rep-like substrate HP(14+14). Molecular weight ladder is shown on Lane 6. Bar graphs with the quantification of covalent complexes are shown below the SDS-PAGE gels. Data showed mean±s.d. of three independent experiments.
Mentions: Relaxases contain either one (Y1) or two (Y2) catalytic tyrosines [1,12]. The ssDNA U-turn observed in Y2 relaxase domains TrwCR- and TraIR -DNA complex structures was also observed in the structure of Y1 relaxase NES [13–15]. However, relevant differences in U-turn formation have been described [12]. In order to check if Y1 relaxases could also catalyze cleavage of Rep-like substrates, we analyzed these substrates for the best known Y1-relaxases, MobA_RSF1010 and TraI_RP4 (S7 Fig). The MobA_RSF1010 binding site was identified as a 10 bp IR that forms a hairpin structure upstream from nic [16]. Mozingo et al. modeled the relaxase minMobA (amino acids 1 to 186, named MobAR in this study) complexed with a 33-mer oligonucleotide and compared this model with TrwCR bound to the 23-mer [16]. According to this comparison, only nucleotides at the base of the 10 bp-hairpin interact with MobAR. To study the influence of IR length, we designed a shorter wt substrate, containing a 6 bp IR WQ(23+7) (Fig 3A and Table 2). Analysis by SDS-PAGE of the covalent complexes obtained after incubation with the protein showed that either the long wt substrate WQ(30+7) (Fig 3A Lane 2) or the short wt substrate WQ(23+7) (Fig 3A Lane 3) generated 40% covalent complexes. These results confirm that just the nucleotides at the base of the stem make specific interactions with relaxase MobAR.

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