Base-flipping mechanism in postmismatch recognition by MutS

Abstract

DNA mismatch recognition and repair is vital for preserving the fidelity of the genome. Conserved across prokaryotes and eukaryotes, MutS is the primary protein that is responsible for recognizing a variety of DNA mismatches. From molecular dynamics simulations of the Escherichia coli MutS-DNA complex, we describe significant conformational dynamics in the DNA surrounding a G·T mismatch that involves weakening of the basepair hydrogen bonding in the basepair adjacent to the mismatch and, in one simulation, complete base opening via the major groove. The energetics of base flipping was further examined with Hamiltonian replica exchange free energy calculations revealing a stable flipped-out state with an initial barrier of ~2 kcal/mol. Furthermore, we observe changes in the local DNA structure as well as in the MutS structure that appear to be correlated with base flipping. Our results suggest a role of base flipping as part of the repair initiation mechanism most likely leading to sliding-clamp formation.

Figure 1

X-ray crystal structure of E. coli MutS (12). (A) MutS is colored with respect to its DNA binding domains (red/pink), connector domains (orange or pale orange), core domains (yellow or pale yellow), clamp domains (green or pale green), and ATPase domains (blue or pale blue). DNA (beige) bases and (brown) backbone. Bound nucleotides are omitted for clarity. (B) A conserved Phe³⁶-X^aa-Glu³⁸ motif interacts with the G·T mismatch through the DNA minor groove. (Green) Protein; (pink) the mismatch; and (yellow) G/C(−1) basepair with the 5′ adjacent base C21. (Black dotted lines) Bifurcated basepair hydrogen bond in the G·T mismatch and hydrogen bonding between Glu³⁸ and T22.

Figure 2

DNA basepair hydrogen bonding for C/G(−2), G/C(−1), G·T mismatch, and A/T(+1) basepairs from N3-N1 (C/G basepairs), N1-N3 (A/T basepairs), and N1-O4 (G·T mismatch) distance time series in each simulation are described here. (Blue dotted lines) Typical hydrogen-bond distances of 3 Å. (A) Wild-type simulations with different nucleotide combinations. (B) F36A mutant simulations.

Figure 3

Correlation of C21 base flipping in NONE:NONE simulation with various structural quantities (see Materials and Methods for definitions): (A) Pseudodihedral angle. (B) C21 backbone ζ-torsion angle. (C) Movement of the S1 DNA binding domain (S1-D1) along X. (D) Movement of S2-D1 along X. (E) Movement of S2-D1 along Y. (F) Movement of S2-D1 along Z. (G) S2 Ser⁶⁶⁸ to S1 Asn⁶¹⁶ C_α-C_α distance. (H) S1 Asn⁶¹⁶ Ψ backbone torsion angle. (I) Salt bridge distance between S2 Arg⁶⁶⁷ and S1 Glu⁵⁹⁴ measured between heavy atoms. (Blue line) A distance of 3 Å corresponding to hydrogen bonding. (J) C_α-RMSD of the S2 signature loop. (K) Snapshots of base-flipping progress viewed from the major groove. Protein, water, and additional DNA are omitted for clarity. (Pink) G·T. (Yellow) G/C(−1). (Gray) C/G(−2). (Red arrow) C21.

Figure 4

Free energy profiles from the HREM simulation: (A) Free energy of base flipping (10.5 ns/replica). (B) C21 backbone ζ-torsion angle versus base flipping. (C) Movement of S1-D1 along X versus base flipping. (D) Movement of S2-D1 along X versus base flipping. (E) Movement of S2-D1 along Y versus base flipping. (F) Movement of S2-D1 along Z versus base flipping. (G) S2 Ser⁶⁶⁸ to S1 Arg⁶¹⁶ C_α-C_α distance versus base flipping. (H) S1 Arg⁶¹⁶ Ψ backbone torsion angle versus base flipping.