MutS switches between two fundamentally distinct clamps during mismatch repair - PubMed (original) (raw)

MutS switches between two fundamentally distinct clamps during mismatch repair

Cherlhyun Jeong et al. Nat Struct Mol Biol. 2011 Mar.

Abstract

Single-molecule trajectory analysis has suggested DNA repair proteins may carry out a one-dimensional (1D) search on naked DNA encompassing >10,000 nucleotides. Organized cellular DNA (chromatin) presents substantial barriers to such lengthy searches. Using dynamic single-molecule fluorescence resonance energy transfer, we determined that the mismatch repair (MMR) initiation protein MutS forms a transient clamp that scans duplex DNA for mismatched nucleotides by 1D diffusion for 1 s (~700 base pairs) while in continuous rotational contact with the DNA. Mismatch identification provokes ATP binding (3 s) that induces distinctly different MutS sliding clamps with unusual stability on DNA (~600 s), which may be released by adjacent single-stranded DNA (ssDNA). These observations suggest that ATP transforms short-lived MutS lesion scanning clamps into highly stable MMR signaling clamps that are capable of competing with chromatin and recruiting MMR machinery, yet are recycled by ssDNA excision tracts.

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Figures

Figure 1

Figure 1. Single–molecule FRET of Taq MutS on duplex DNA

(a) The crystal structure of homodimer Taq MutS bound to unpaired dT (PDB:1EWQ). Donor Cy3 was conjugated to C469 of Taq MutS(C42A, T469C). (b) Schematic representation of smFRET assay. Cy5–labeled matched DNA molecules (74 bp) were immobilized on a quartz surface via a biotin–streptavidin linker and the open–end blocked antidigoxigenin. (c) Representative traces of fluorescent intensity and FRET efficiency for matched DNA molecules in the presence of 10 nM MutS and 100 mM KCl. (d) The distributions of binding lifetime and dissociation time for 10 nM MutS in 100 mM KCl. A single exponential with mean ± s.e.m. fit the distribution.

Figure 2

Figure 2. Taq MutS scans duplex DNA by rotational diffusion

(a) FRET efficiency determined from a population histogram of 74 bp duplex DNA molecules at a 30 ms time resolution. (b) FRET efficiency determined from a population histogram of 100 bp duplex DNA molecules at a 30 ms time resolution and 4 ms time resolution, respectively. (c) Averaged FRET value from individual traces of MutS diffusion on duplex DNA was determined. The Gaussian distribution of the FRET efficiency resulted in a refined FRET efficiency of 0.481 ± 0.023 (mean ± s.d.) on the 74 bp duplex DNA (n = 78) and 0.341 ± 0.031 (mean ± s.d.) on the 100 bp duplex DNA (n = 89) (left panel). For the transitional diffusion model, the circumference distance between Cy3 and Cy5 determined by the random initial binding remains constant during MutS diffusion. The distributions of FRET efficiency were obtained from 100 trials in silico of the arbitrary binding position of MutS for the 74 bp and 100 bp duplex DNAs, which evenly range from 0.291 to 0.655 and 0.199 to 0.443, respectively (right panel). In contrast, the circumference distance varies with rotational diffusion in the rotational diffusion model (middle panel). The resulting distribution of FRET values displays a Gaussian with a sharp peak (0.479 ± 0.007 for 74 bp; 0.322 ± 0.011 for 100 bp). The errors indicate s.d. (d) The dissociation constant with blocked ends was determined from the intercept of _τ_duplex·on and_τ_duplex·off for MutS binding to duplex DNA. (e) The dissociation constant with an open–ended DNA was determined from the intercept of _τ_duplex·on and_τ_duplex·off for MutS binding to 3′ –unblocked duplex DNA.

Figure 3

Figure 3. Single–molecule FRET of Taq MutS binding to a +dT mismatch

(a) Schematic illustration of mismatched DNA molecules containing a single unpaired +dT. Cy5 is positioned at the ninth base from the +dT mismatch. Yellow indicates nucleotides that contact MutS residues during mismatch binding–. (b) Representative traces of fluorescence intensity and FRET value for a +dT mismatch in the presence of 10 nM MutS and 100 mM KCl. (c) FRET efficiency when MutS was bound to the +dT mismatch. (d) The distributions of MutS binding lifetime and dissociation time for 10 nM MutS in 100 mM KCl. A single exponential with mean ± s.e.m. fit the distribution. (e) On–rate (k+dT·on = 1/τ+dT·off) and off–rate (k+dT·off = 1/τ+dT·on) vs. concentration of Taq MutS. The dissociation constant was determined at the intercept of _τ_duplex·on and_τ_duplex·off for MutS binding to a +dT mismatch. (f) Representative trace of fluorescent intensity and FRET value showing the searching kinetics followed by binding kinetics for a +dT mismatch. (g) FRET efficiency when MutS is searching for a mismatch. (h) The distributions of binding lifetime for MutS in search of a mismatch at a 30 ms time resolution. (i) The distributions of binding lifetime for MutS in search of a mismatch at a 4 ms time resolution. (j) The frequency of single molecules where MutS is found searching for a mismatch at a 30 ms and 4 ms time resolution.

Figure 4

Figure 4. ATP induces an exceptionally long–lived FRET state of MutS

(a) Schematic representation of ATP/ATPγS effects on mismatch bound MutS and Representative traces of fluorescence intensity and FRET value for a +dT mismatch in the presence of 10 nM MutS, 100 mM KCl and 200 μM ATP. (b) High FRET efficiency and intermediate FRET efficiency determined from a population histogram of +dT mismatched DNA molecules. (c) The distributions of MutS binding lifetime to a +dT mismatch in the presence of 1 mM ATP. (d) Representative time–lapse trace of ATP–bound Taq MutS on the +dT mismatched DNA substrate. (e) Dwell time of the intermediate FRET state of MutS in the presence of ATP and ATPγS determined from a single exponential of a population histogram of +dT molecules. (f) The frequency of one (yellow), two (green), and three (blue) MutS sliding clamps found on 100 bp and 74 bp single DNA molecules in the presence of 30 nM or 300 nM MutS.

Figure 5

Figure 5. Single–stranded DNA provokes the release of ATP–bound MutS Sliding Clamps

(a) Representative traces of fluorescence intensity and FRET value for a +dT mismatch containing a (dT)10 single–stranded DNA 5′–tail in the presence of 10 nM MutS, 100 mM KCl, and 200 μM ATP. Schematic representation of +dT mismatch containing a (dT)10 single–stranded DNA 5′–tail with MutS and ATP/ATPγS. (b) The distributions of FRET efficiency and binding lifetime determined from a population histogram of +dT–(dT)10 DNA molecules. (c) The distributions of FRET efficiency and binding lifetime determined from a population histogram of +dT–(dT)10 DNA molecules in the presence of 200 μM ATP. (d) The distributions of binding lifetime determined from a histogram of +dT–(dT)10 DNA molecules in the presence of 200 μM ATPγS.

Figure 6

Figure 6. The role of distinct MutS clamps in the Molecular Switch Model for MMR

(a) MutS searching clamps. (b) MutS mismatch binding and sliding clamps. (c) MutS–MutL complexes with an MMR excision tract that provokes the release of MutS sliding clamps.

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References

    1. Kolodner RD, Marsischky GT. Eukaryotic DNA mismatch repair. Current Opin Genet Dev. 1999;9:89–96. - PubMed
    1. Modrich P, Lahue R. Mismatch repair in replication fidelity, genetic recombination, and cancer biology. Annu Rev Biochem. 1996;65:101–33. - PubMed
    1. Kolodner RD, Mendillo ML, Putnam CD. Coupling distant sites in DNA during DNA mismatch repair. Proc Natl Acad Sci U S A. 2007;104:12953–4. - PMC - PubMed
    1. Acharya S, Foster PL, Brooks P, Fishel R. The coordinated functions of the E. coli MutS and MutL proteins in mismatch repair. Mol Cell. 2003;12:233–46. - PubMed
    1. Gradia S, Acharya S, Fishel R. The role of mismatched nucleotides in activating the hMSH2–hMSH6 molecular switch. J Biol Chem. 2000;275:3922–3930. - PubMed

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