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. 2018 Aug 21;46(14):7193-7205.
doi: 10.1093/nar/gky530.

The Mitochondrial Single-Stranded DNA Binding Protein From S. Cerevisiae, Rim1, Does Not Form Stable Homo-Tetramers and Binds DNA as a Dimer of Dimers

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The Mitochondrial Single-Stranded DNA Binding Protein From S. Cerevisiae, Rim1, Does Not Form Stable Homo-Tetramers and Binds DNA as a Dimer of Dimers

Saurabh P Singh et al. Nucleic Acids Res. .
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Abstract

Rim1 is the mitochondrial single-stranded DNA binding protein in Saccharomyces cerevisiae and functions to coordinate replication and maintenance of mtDNA. Rim1 can form homo-tetramers in solution and this species has been assumed to be solely responsible for ssDNA binding. We solved structures of tetrameric Rim1 in two crystals forms which differ in the relative orientation of the dimers within the tetramer. In testing whether the different arrangement of the dimers was due to formation of unstable tetramers, we discovered that while Rim1 forms tetramers at high protein concentration, it dissociates into a smaller oligomeric species at low protein concentrations. A single point mutation at the dimer-dimer interface generates stable dimers and provides support for a dimer-tetramer oligomerization model. The presence of Rim1 dimers in solution becomes evident in DNA binding studies using short ssDNA substrates. However, binding of the first Rim1 dimer is followed by binding of a second dimer, whose affinity depends on the length of the ssDNA. We propose a model where binding of DNA to a dimer of Rim1 induces tetramerization, modulated by the ability of the second dimer to interact with ssDNA.

Figures

Figure 1.
Figure 1.
Crystal structures of Rim1. (A, B) Models of Rim1 tetramers determined from crystal Form 1 in A and 2 in B. (C, D) Zoom-in of the dimer–dimer interfaces highlighting the potential salt-bridges (dash lines) in crystal Form 1 in C and Form 2 in D. (E) Model of a Rim1 monomer. (F) Superimposition of all the Rim1 monomers from crystal Form 1 and 2. (G) Superimposition of the monomer in E with monomers from EcSSB (PDB: 1sru, cyan), Plasmodium falciparum SSB (PDB: 3ulp, pink), and HsmtSSB (PDB: 3ull, yellow). (H) Position of Y61 of Rim1 in crystal Form 1 and 2 relative to the histidine of EcSSB, PfSSB and HsmtSSB.
Figure 2.
Figure 2.
Rim1 does not form stable tetramers in solution. (A) The sedimentation coefficient as a function of Rim1 concentration (monomers) in Buffer HK150, for two preparations of the protein (• and ▴) and in Buffer HK1000 (♦). The solid black line is the fit of the data collected in Buffer HK150 to a dimer–tetramer oligomerization model (equations s1 and s4 in Supplementary material) with L0 = (2 ± 0.3) x 105 M−1 and sT = (3.9 ± 0.1) S, keeping sD fixed at 2.1 S. The protein concentration dependences for HsmtSSB and Rim1Y85H are shown in blue and red, respectively. The dashed lines are linear fits meant to indicate little protein concentration dependence of the s20,w. (B) Protein concentration dependence of the molecular weight of Rim1 in Buffers HK150 (•), HK150M5 (formula image), HK20 (▪) and HK1000 (♦). The data in red are for Rim1Y85H. The solid black line is a simulation with a dimer–tetramer with L0 = 5 × 105 M−1, meant to capture the trend of the data.
Figure 3.
Figure 3.
Rim1 binds ssDNA with an occluded site-size of ∼14 nt per monomer. (A) Quenching of tryptophan fluorescence as a function of the ratio of the concentration of poly (dT) (nt) to the concentration of Rim1 (monomers), in Buffers HK150 (•) and HK1 (▪). (B) Occluded site-size in nucleotides per Rim1 monomer as a function of KCl concentration. (C) Stoichiometry of Rim1-DNA complexes (•) formed with different lengths of ssDNA in Buffer HK150, under conditions where Rim1 is predominantly a tetramer. In red are the data for Rim1Y85H. (D) Stoichiometry of Rim1-DNA complexes formed in Buffer HK150 with dT15-Cy3-T (▪) and dT20-Cy3-T (formula image), as a function of Rim1 concentration. In red (solid and open symbol) are the corresponding data for Rim1Y85H.
Figure 4.
Figure 4.
On short ssDNA high-affinity binding of one Rim1 dimer is accompanied by lower affinity binding of a second one. (A) The left panel shows the change in fluorescence anisotropy (circles) and total intensity (squares) of FAM-dT20 at different concentrations (50 nM black, 100 nM blue, 200 nM red, 400 nM grey) as a function of Rim1 concentration (dimer), in Buffer HK150M5. The solid lines are the fits with a 2:1 binding model (equations s6 and s10 in Supplementary material). The right panel shows the dependence of the monitored signals as a function of the degree of binding calculated from the data in the left panel. The lines indicate the estimated values for different signals corresponding to one and two ligand-bound states and their associated stoichiometry. (B) Same as in A but for Rim1Y85H and the change in fluorescence anisotropy.
Figure 5.
Figure 5.
The affinity of the second Rim1 dimer is modulated by the length of ssDNA available for interaction. (A) Change in fluorescence intensity of dT38-Cy3-T as a function of the ratio of Rim1 concentration to the concentration of the DNA (200 nM), in Buffer HK150 (•) and HK150M5 (formula image). (B) Same as in A but for Cy5.5-dT40-Cy3-T, monitoring the change in Cy5.5 fluorescence intensity upon excitation of Cy3. (C) The change in fluorescence anisotropy (circles) and total intensity (squares) of dT38-FL as a function of Rim1 concentration (monomer) to the concentration of the DNA (200 nM), in Buffer HK150 (•) and HK150M5 (formula image). (D) The change in fluorescence anisotropy (circles) and total intensity (squares) of dT38-FL at different concentrations (50 nM red, 100 nM blue, 200 nM gray, 400 nM black) as a function of Rim1 concentration (dimer), in Buffer HK150M5. The solid lines are the fits with a 2:1 binding model (equations s6 and s10 in Supplementary material). (E) Same as in D but in Buffer HK1000M5. (F) Same as in D but for Rim1Y85H. (G, H) Same as in A and B but for dT59-Cy3-T in G and Cy5-dT68-Cy3-T in H. (I) Analysis with a 1:1 binding model (Supplementary material) with different binding constants (1 × 109 M−1 blue, 1 × 1010 M−1 black, and 1 × 1011 M−1 red) and assuming that in Buffer HK1000M5 Rim1 binds to dT59-Cy3-T (50 nM) as a preformed tetramer.
Figure 6.
Figure 6.
Working model showing how DNA binding is coupled to the oligomeric state of Rim1. Rim1 exists in solution in a dimer–tetramer equilibrium with L0 intrinsic oligomerization constant. In the left branch of the model, Rim1 binds ssDNA as a pre-formed tetramer with an equilibrium binding constant KT that is currently undetermined. The models for ssDNA bound to Rim1 were based on the DNA in the crystal structure of EcSSB (PDB:1eyg). The right branch of the model shows binding of two dimers of Rim1 and possible protein–DNA complexes (A-D) that can be formed depending of the length of the ssDNA. Independent of the ssDNA length, the binding constant of the second Rim1 dimer, LN,m, is larger than L0, indicating that DNA binding to the first Rim1 dimer favors tetramerization. Also, for N ≈ 4 m LN,m increases to the point that it cannot be distinguished from KD, and Rim1 appears to bind as a pre-formed tetramer.

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