The effects of nucleotides on MutS-DNA binding kinetics clarify the role of MutS ATPase activity in mismatch repair - PubMed (original) (raw)

The effects of nucleotides on MutS-DNA binding kinetics clarify the role of MutS ATPase activity in mismatch repair

Emily Jacobs-Palmer et al. J Mol Biol. 2007.

Abstract

MutS protein initiates mismatch repair with recognition of a non-Watson-Crick base-pair or base insertion/deletion site in DNA, and its interactions with DNA are modulated by ATPase activity. Here, we present a kinetic analysis of these interactions, including the effects of ATP binding and hydrolysis, reported directly from the mismatch site by 2-aminopurine fluorescence. When free of nucleotides, the Thermus aquaticus MutS dimer binds a mismatch rapidly (k(ON)=3 x 10(6) M(-1) s(-1)) and forms a stable complex with a half-life of 10 s (k(OFF)=0.07 s(-1)). When one or both nucleotide-binding sites on the MutS*mismatch complex are occupied by ATP, the complex remains fairly stable, with a half-life of 5-7 s (k(OFF)=0.1-0.14 s(-1)), although MutS(ATP) becomes incapable of (re-)binding the mismatch. When one or both nucleotide-binding sites on the MutS dimer are occupied by ADP, the MutS*mismatch complex forms rapidly (k(ON)=7.3 x 10(6) M(-1) s(-1)) and also dissociates rapidly, with a half-life of 0.4 s (k(OFF)=1.7 s(-1)). Integration of these MutS DNA-binding kinetics with previously described ATPase kinetics reveals that: (a) in the absence of a mismatch, MutS in the ADP-bound form engages in highly dynamic interactions with DNA, perhaps probing base-pairs for errors; (b) in the presence of a mismatch, MutS stabilized in the ATP-bound form releases the mismatch slowly, perhaps allowing for onsite interactions with downstream repair proteins; (c) ATP-bound MutS then moves off the mismatch, perhaps as a mobile clamp facilitating repair reactions at distant sites on DNA, until ATP is hydrolyzed (or dissociates) and the protein turns over.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Selective interaction of MutS with +T mismatched DNA. (A) T. aquaticus MutS Phe 39 with +T DNA with 2-AP positioned 3′ to the +T insertion. (B) Emission spectra of 2-AP:AT and 2-AP:+T DNA in the absence (○, □) and presence (●, ■) of MutS upon excitation at 315 nm show increase in fluorescence when MutS binds +T DNA. (C) Titrations of 10 nM 2-AP:AT (●) and 2-AP:+T (■) with 0 – 0.2 μM MutS, and 2-AP:+T (◇) with MutS F39A reveal interaction only between wildtype MutS and + T DNA (_K_D = 15 ± 2.5 nM for MutS•+T complex). (D) Anisotropy measurements of 10 nM TAMRA-labeled AT (●) and +T DNA (■) titrated with 0 – 0.2 μM MutS, and +T DNA (◇) with MutS F39A confirm high-affinity binding of MutS to mismatched DNA (_K_D = 25 ± 1.7 nM for MutS•+T complex). (E) 2-AP:+T suppresses ATP hydrolysis by MutS (1 μM dimer) from 9.2 ± 0.8 s−1 (no DNA or AT DNA) to 0.3 s−1, while MutS F39A ATPase activity remains unaffected by +T DNA (1 μM MutS dimer, 500 μM ATP, 3 μM DNA)

Figure 2

Figure 2

MutS binds +T DNA rapidly to form a stable complex. (A) Stopped-flow traces of 30 nM 2-AP-labeled DNA mixed with 200 nM MutS reveal rapid binding of wildtype MutS to +T DNA (_k_observed = 0.53 ± 0.005 s−1), but no interaction between MutS and AT DNA or MutS F39A and +T DNA. (B) MutS concentration dependence of _k_observed yields _k_ON = 3 ± 0.2 x 106 M−1 s−1. (C) A pre-formed complex of 2-AP:+T (30 nM) and MutS (200 nM) mixed with 4 μM unlabeled +T DNA trap yields _k_OFF = 0.07 ± 0.0003 s−1 for MutS•+T complex.

Figure 3

Figure 3

Slow ATP-induced loss of MutS interaction with a mismatch in DNA. (A) Increasing concentrations of ATPγS (0 – 60 μM) result in loss of 2-AP:+T (30 nM) and MutS (200 nM) binding. (B) A similar result is obtained with ATP hydrolysis-deficient MutS E663A with increasing ATP. (C) A plot of the changing amplitude versus ATPγS or ATP concentration fit to a hyperbola yields _K_1/2 = 1.7 ± 0.5 μM or 0.8 ± 0.1 μM, respectively, for ATPγS/ATP effects on MutS. (D) Anisotropy measurements of 10 nM TAMRA:+T DNA and 200 nM MutS titrated with 0–40 μM ATPγS confirm the loss of binding and yield _K_1/2 = 0.7 ± 0.1 μM for the nucleotide effect on MutS. (E) A pre-formed complex of 2-AP:+T (30 nM) and MutS (200 nM) mixed with 4 μM unlabeled +T DNA trap and 150 μM ATPγS or ATP yields _k_OFF = 0.14 ± 0.001 s−1 and 0.1 ± 0.001 s−1 for MutS•+T and MutS E663A•+T complexes, respectively.

Figure 4

Figure 4

ADP-induced rapid DNA binding and release by MutS. (A) Presence of ADP (0–60 μM) in the reaction with 30 nM 2-AP:+T and 200 nM MutS results in a decrease in amplitude and an apparent increase in the rate of MutS binding to +T DNA. (B) A plot of the changing amplitude versus ADP concentration fit to a hyperbola reveals a new, lower equilibrium concentration of MutS•+T complex (~ 50 %), and yields _K_1/2 = 2.4 ± 0.6 μM for the ADP effect on MutS. (C) Anisotropy measurements of 10 nM TAMRA:+T DNA and 200 nM MutS titrated with 0–80 μM ADP confirm the decrease in MutS•+T complex and yield _K_1/2 = 1.3 ± 0.4 μM for the ADP effect on MutS. (D) A pre-formed complex of 2-AP:+T (30 nM) and MutS (200 nM) mixed with 4 μM unlabeled +T DNA trap and 150 μM ADP yields _k_OFF = 1.74 ± 0.03 s−1 for MutS•+T.

Figure 5

Figure 5

Implications of the nucleotide effects on MutS-DNA interaction in mismatch repair. (A) The effect of ATP on MutSADP•+T was tested by mixing pre-incubated 30 nM 2-AP:+T, 200 nM MutS, and 8 μM ADP with 4 μM +T DNA trap and 150 μM ADP (■) or 150 μM ATPγS (◇), which yielded _k_OFF = 1.56 ± 0.07 s−1 and 0.17 ± 0.003 s−1 , respectively, indicating that ATP rapidly replaces ADP on MutS and stabilizes MutS•+T complex. (B) In the absence of DNA (a), MutSFREE can bind ATP (b), hydrolyze it and release Pi rapidly (*only the activity of the rapid ATP-hydrolyzing subunit is shown in this model) to form MutSADP (c). When MutSFREE binds mismatched DNA, rapidly and with high affinity (d), ATP binding is fast but ATP hydrolysis is suppressed and MutSATP, which forms a stable complex with the mismatch (e), becomes predominant. MutSADP also binds mismatched DNA rapidly (f), but the interaction is unstable. However, the ADP can be replaced readily by ATP and mismatch-bound MutSATP (e) is again predominant. That both pathways lead to the MutSATP•mismatch complex suggests a key role for this species in the repair reaction. In time, MutSATP releases the mismatch (b, g), in a conformation that precludes rebinding until ATP dissociates or is hydrolyzed.

Similar articles

Cited by

References

    1. Peltomaki P. Role of DNA mismatch repair defects in the pathogenesis of human cancer. J Clin Oncol. 2003;21:1174–9. - PubMed
    1. Li GM. DNA mismatch repair and cancer. Front Biosci. 2003;8:d997–1017. - PubMed
    1. Iyer RR, Pluciennik A, Burdett V, Modrich PL. DNA mismatch repair: functions and mechanisms. Chem Rev. 2006;106:302–23. - PubMed
    1. Kunkel TA, Erie DA. DNA Mismatch Repair. Annu Rev Biochem. 2005;74:681–710. - PubMed
    1. Schofield MJ, Hsieh P. DNA mismatch repair: molecular mechanisms and biological function. Annu Rev Microbiol. 2003;57:579–608. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources