Saccharomyces cerevisiae Msh2-Msh6 DNA binding kinetics reveal a mechanism of targeting sites for DNA mismatch repair

Abstract

The DNA mismatch repair system (MMR) identifies replication errors and damaged bases in DNA and functions to preserve genomic integrity. MutS performs the task of locating mismatched base pairs, loops and lesions and initiating MMR, and the fundamental question of how this protein targets specific sites in DNA is unresolved. To address this question, we examined the interactions between Saccharomyces cerevisiae Msh2-Msh6, a eukaryotic MutS homolog, and DNA in real time. The reaction kinetics reveal that Msh2-Msh6 binds a variety of sites at similarly fast rates (k (ON) approximately 10(7) M(-1) s(-1)), and its selectivity manifests in differential dissociation rates; e.g., the protein releases a 2-Aminopurine:T base pair approximately 90-fold faster than a G:T mismatch. On releasing the 2-Ap:T site, Msh2-Msh6 is able to move laterally on DNA to locate a nearby G:T site. The long-lived Msh2-Msh6.G:T complex triggers the next step in MMR--formation of an ATP-bound clamp--more effectively than the short-lived Msh2-Msh6.2-Ap:T complex. Mutation of Glu in the conserved Phe-X-Glu DNA binding motif stabilizes Msh2-Msh6(E339A).2-Ap:T complex, and the mutant can signal 2-Ap:T repair as effectively as wild-type Msh2-Msh6 signals G:T repair. These findings suggest a targeting mechanism whereby Msh2-Msh6 scans DNA, interrogating base pairs by transient contacts and pausing at potential target sites, and the longer the pause the greater the likelihood of MMR.

Fig. 1.

Msh2-Msh6 binds MMR targets rapidly and with varying affinities. (A) Equilibrium fluorescence anisotropy measurements with TAMRA (green dot in scheme) end-labeled DNAs yield K_D = 4.4 ± 0.5, 3.3 ± 0.5, and 28 ± 1.6 nM for G:T_TAMRA (dark blue), O⁶MeG:T_TAMRA (red), and +T_TAMRA (green), respectively, and almost no binding to G:C_TAMRA (light blue). (B) Kinetic measurements after mixing Msh2-Msh6 with DNA yield k_ON = 0.95, 3, and 1.7 × 10⁷ M^-1 s^-1, respectively, for the three DNAs, and no detectable binding to G:C_TAMRA. (C) Mixing Msh2-Msh6·DNA_TAMRA complexes with unlabeled G:T trap yields k_OFF = 0.013, 0.005, and 0.15 s^-1, respectively. (D) Free Msh2-Msh6 catalyzes a burst of ATP hydrolysis and Pi release (k_burst = 1.2 s^-1, k_cat = 0.18 s^-1) and the DNAs inhibit ATP hydrolysis to varying degrees.

Fig. 2.

An on-site 2-Ap reporter confirms that Msh2-Msh6 dissociation rates underlie target site selectivity. (A) Equilibrium 2-Ap fluorescence measurements yield K_D = 13 ± 2 nM for G:T_2-Ap (dark blue) and 70 ± 7 nM for G:C_2-Ap DNA (light blue); 2-Ap:T (purple dot) is located next to G:T or G:C, respectively. (B) Kinetic measurements yield k_ON = 2.4 and 1.5 × 10⁷ M^-1 s^-1 and (C) k_OFF = 0.013 and 1.2 s^-1 for G:T_2-Ap and G:C_2-Ap, respectively. (D) ATP hydrolysis is partially suppressed by G:C_2-Ap DNA.

Fig. 3.

Msh2-Msh6 moves from 2-Ap:T to G:T without dissociating from DNA. (A) Fluorescence anisotropy shows Msh2-Msh6 binding mismatched 2-Ap:T/7 bp/G:T_TAMRA and matched 2-Ap:T/7 bp/G:C_TAMRA DNA rapidly (k_ON = 1.35 and 1.4 × 10⁷ M^-1 s^-1, respectively). (B) 2-Ap fluorescence yields the same rate for 2-Ap:T/7 bp/G:C_TAMRA, but 2-Ap:T/7 bp/G:T_TAMRA shows biphasic kinetics. (C) 2-Ap fluorescence reports rapid release of Msh2-Msh6 from 2-Ap:T in 2-Ap:T/7 bp/G:T_TAMRA with an external trap (unlabeled G:T) or with only buffer added after 0.25 s (k_OFF = 0.8–1.2 s^-1). (D) Corresponding TAMRA anisotropy shows slow release of Msh2-Msh6 from 2-Ap:T/7 bp/G:T_TAMRA (k_OFF = 0.044 s^-1).