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The RdgC protein employs a novel mechanism involving a finger domain to bind to circular DNA

Briggs, G. S., Yu, J., Mahdi, A. A., Lloyd, R. G.·2010·Nucleic Acids Research
DOI 10.1093/nar/gkq509PMC PMC2965237
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Figure 1. Structure of RdgC. (PDB co-ordinates 2OWL). (A) A ribbon diagram showing the ring structure of RdgC and identifying the horseshoe and gate regions. The dotted line indicates the horseshoe domain interface. (B) A rotated ribbon diagram illustrating projection of the finger domains. The dotted line indicates the gate domain interface. (C) Detail of one of the hydrogen bond networks at the horseshoe interface between the Q212 and E218 residues on one dimer and K227 on the other. The conserved H222 residues are also shown. (D) Detail of the gate interface interactions, showing the conserved F120 residues and the modelled disulphide formed in the R118C derivative.
Figure 1. Structure of RdgC. (PDB co-ordinates 2OWL). (A) A ribbon diagram showing the ring structure of RdgC and identifying the horseshoe and gate regions. The dotted line indicates the horseshoe domain interface. (B) A rotated ribbon diagram illustrating projection of the finger domains. The dotted line indicates the gate domain interface. (C) Detail of one of the hydrogen bond networks at the horseshoe interface between the Q212 and E218 residues on one dimer and K227 on the other. The conserved H222 residues are also shown. (D) Detail of the gate interface interactions, showing the conserved F120 residues and the modelled disulphide formed in the R118C derivative.CC BY-NC · PubMed Central
Figure 2. The effect of ΔrdgC on the viability of ΔpriA and ΔpriA dnaC810 strains. Photographs of plates illustrating the results of synthetic lethality assays. All constructs contained a priA+ derivative of the pRC7 plasmid, and white colonies indicate the segregation of plasmid-free cells. Cells were plated on either LB agar (rich medium) or MA (56/2 minimal agar medium) as indicated.
Figure 2. The effect of ΔrdgC on the viability of ΔpriA and ΔpriA dnaC810 strains. Photographs of plates illustrating the results of synthetic lethality assays. All constructs contained a priA+ derivative of the pRC7 plasmid, and white colonies indicate the segregation of plasmid-free cells. Cells were plated on either LB agar (rich medium) or MA (56/2 minimal agar medium) as indicated.CC BY-NC · PubMed Central
Figure 3. Analytical gel filtration analysis of RdgC proteins. (A) Gel filtration of purified WT RdgC protein. Protein was diluted to a final concentration of 10 μM (dimer) in gel filtration buffer and either analysed immediately or incubated overnight at 4°C before analysis on a Superdex 75 10/300 column (GE Healthcare). (B) Gel filtration of purified RdgC proteins. Protein was diluted to a final concentration of 10 μM (dimer) in gel filtration buffer and incubated overnight at 4°C before analysis on a Superdex 75 10/300 column (GE Healthcare). (C) Gel filtration of purified RdgC R118C F120T protein. Protein was diluted to a final concentration of 10 μM (dimer) in gel filtration buffer and either analysed immediately or incubated overnight at 4°C before analysis on a Superdex 75 10/300 column (GE Healthcare). An additional sample was incubated and analysed in gel filtration buffer containing 5 mM DTT. (D) Gel filtration of purified RdgC Δfinger proteins. Proteins were diluted to a final concentration of 10 μM (dimer) in gel filtration buffer and incubated overnight at 4°C before analysis on a Superdex 75 10/300 column (GE Healthcare). An additional sample of Δfinger R118C was incubated and analysed in gel filtration buffer containing 5 mM DTT.
Figure 3. Analytical gel filtration analysis of RdgC proteins. (A) Gel filtration of purified WT RdgC protein. Protein was diluted to a final concentration of 10 μM (dimer) in gel filtration buffer and either analysed immediately or incubated overnight at 4°C before analysis on a Superdex 75 10/300 column (GE Healthcare). (B) Gel filtration of purified RdgC proteins. Protein was diluted to a final concentration of 10 μM (dimer) in gel filtration buffer and incubated overnight at 4°C before analysis on a Superdex 75 10/300 column (GE Healthcare). (C) Gel filtration of purified RdgC R118C F120T protein. Protein was diluted to a final concentration of 10 μM (dimer) in gel filtration buffer and either analysed immediately or incubated overnight at 4°C before analysis on a Superdex 75 10/300 column (GE Healthcare). An additional sample was incubated and analysed in gel filtration buffer containing 5 mM DTT. (D) Gel filtration of purified RdgC Δfinger proteins. Proteins were diluted to a final concentration of 10 μM (dimer) in gel filtration buffer and incubated overnight at 4°C before analysis on a Superdex 75 10/300 column (GE Healthcare). An additional sample of Δfinger R118C was incubated and analysed in gel filtration buffer containing 5 mM DTT.CC BY-NC · PubMed Central
Figure 4. Oligonucleotide binding by RdgC proteins. (A) Representative band-shift assays with dsDNA. Reactions used the RdgC proteins indicated at 0, 0.375, 0.75, 1.5, 3, 6, 12, 24 and 1850 nM (of dimer) and 32P-labelled RGL13/17 DNA at 0.2 nM. Assays with R118A, R118C, Q212A and H222A gave the same band pattern as the WT protein. Assays with F120S and K227A gave the same band pattern as F120T. (B) Quantification of dsDNA binding. RGL13/17 DNA was used at 0.2 nM. Data are means of at least two band-shift experiments. (C) Quantification of ssDNA binding. 32P-labelled RGL13 DNA was used at 0.2 nM. Data are means of at least two band-shift experiments. (D) Effect of DTT on duplex binding by RdgC R118C F120T. Reactions used mutant RdgC protein at 0, 1, 5, 25, 125 and 625 nM (of dimer) and 32P-labelled RGL13/17 DNA at 0.2 nM. (E) Plasmid binding by RdgC proteins. Reactions used the RdgC proteins indicated at 0, 34, 68, 136, 272, 544, 1088, 2176 and 23 400 nM (of dimer) and pGEM-7 Zf(+) circular dsDNA at 2.2 nM plasmid (6480 nM nucleotide pairs). (F) Plasmid binding by the F120T K227A double mutant. Reactions used mutant RdgC at 0, 1000, 2000, 5000, 10 000 and 20 000 nM (of dimer) and pGEM-7 Zf(+) circular dsDNA at 2.2 nM plasmid (6480 nM nucleotide pairs).
Figure 4. Oligonucleotide binding by RdgC proteins. (A) Representative band-shift assays with dsDNA. Reactions used the RdgC proteins indicated at 0, 0.375, 0.75, 1.5, 3, 6, 12, 24 and 1850 nM (of dimer) and 32P-labelled RGL13/17 DNA at 0.2 nM. Assays with R118A, R118C, Q212A and H222A gave the same band pattern as the WT protein. Assays with F120S and K227A gave the same band pattern as F120T. (B) Quantification of dsDNA binding. RGL13/17 DNA was used at 0.2 nM. Data are means of at least two band-shift experiments. (C) Quantification of ssDNA binding. 32P-labelled RGL13 DNA was used at 0.2 nM. Data are means of at least two band-shift experiments. (D) Effect of DTT on duplex binding by RdgC R118C F120T. Reactions used mutant RdgC protein at 0, 1, 5, 25, 125 and 625 nM (of dimer) and 32P-labelled RGL13/17 DNA at 0.2 nM. (E) Plasmid binding by RdgC proteins. Reactions used the RdgC proteins indicated at 0, 34, 68, 136, 272, 544, 1088, 2176 and 23 400 nM (of dimer) and pGEM-7 Zf(+) circular dsDNA at 2.2 nM plasmid (6480 nM nucleotide pairs). (F) Plasmid binding by the F120T K227A double mutant. Reactions used mutant RdgC at 0, 1000, 2000, 5000, 10 000 and 20 000 nM (of dimer) and pGEM-7 Zf(+) circular dsDNA at 2.2 nM plasmid (6480 nM nucleotide pairs).CC BY-NC · PubMed Central
Figure 5. The effect of rdgC mutations on the viability of ΔpriA and ΔpriA dnaC810 strains. Photographs of plates illustrating the results of synthetic lethality assays. All constructs contained a priA+ derivative of the pRC7 plasmid, and white colonies indicate the segregation of plasmid-free cells. Colonies were grown on either LB agar or MA as indicated.
Figure 5. The effect of rdgC mutations on the viability of ΔpriA and ΔpriA dnaC810 strains. Photographs of plates illustrating the results of synthetic lethality assays. All constructs contained a priA+ derivative of the pRC7 plasmid, and white colonies indicate the segregation of plasmid-free cells. Colonies were grown on either LB agar or MA as indicated.CC BY-NC · PubMed Central
Figure 6. Properties of the finger domain. (A) Electrostatic surface representation of a finger domain, illustrating its electropositive nature and the position of the four conserved basic residues modified in the RdgCfingertip derivative. (B) Synthetic lethality assays showing the effect of finger domain mutations on ΔpriA dnaC810 strain viability. Colonies were grown on LB agar. (C) Quantification of dsDNA binding. Reactions used the RdgC proteins indicated and 32P-labelled RGL13/17 DNA at 0.2 nM. Data are means of at least two band-shift assays. (D) Quantification of ssDNA binding. Reactions used the RdgC proteins indicated and 32P-labelled RGL13 DNA at 0.2 nM. Data are means of at least two band-shift assays. (E) Plasmid binding. Reactions used the RdgC proteins indicated at 0, 34, 68, 136, 272, 544, 1088, 2176 and 23 400 nM (of dimer) and pGEM-7 Zf(+) DNA at 2.2 nM plasmid.
Figure 6. Properties of the finger domain. (A) Electrostatic surface representation of a finger domain, illustrating its electropositive nature and the position of the four conserved basic residues modified in the RdgCfingertip derivative. (B) Synthetic lethality assays showing the effect of finger domain mutations on ΔpriA dnaC810 strain viability. Colonies were grown on LB agar. (C) Quantification of dsDNA binding. Reactions used the RdgC proteins indicated and 32P-labelled RGL13/17 DNA at 0.2 nM. Data are means of at least two band-shift assays. (D) Quantification of ssDNA binding. Reactions used the RdgC proteins indicated and 32P-labelled RGL13 DNA at 0.2 nM. Data are means of at least two band-shift assays. (E) Plasmid binding. Reactions used the RdgC proteins indicated at 0, 34, 68, 136, 272, 544, 1088, 2176 and 23 400 nM (of dimer) and pGEM-7 Zf(+) DNA at 2.2 nM plasmid.CC BY-NC · PubMed Central
Figure 7. Model illustrating how RdgC may load onto DNA. (A) DNA binds across an exposed positive face, interacting with the finger domain. (B) The finger domain rotates, disrupting the gate dimer interface. (C) The gate opens, and RdgC slides over the DNA. (D) The gate closes, encircling the DNA within the ring.
Figure 7. Model illustrating how RdgC may load onto DNA. (A) DNA binds across an exposed positive face, interacting with the finger domain. (B) The finger domain rotates, disrupting the gate dimer interface. (C) The gate opens, and RdgC slides over the DNA. (D) The gate closes, encircling the DNA within the ring.CC BY-NC · PubMed Central

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Metadata

Authors
Briggs, G. S., Yu, J., Mahdi, A. A., & Lloyd, R. G.
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2010-06-04
Volume
38
Issue
19
Pages
6433-6446
ISSN
  • 0305-1048
  • 1362-4962
DOI
10.1093/nar/gkq509
PMC
PMC2965237
Cited by
13
References
0
Language
en
Licence
creativecommons.org/licenses/by-nc/2.5 (vor, 2348 d)

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