DNA Base Flipping: New-Found Insights into the DNA Mismatch Recognition Process in E. Coli Muts

Abstract

Detection of various base-base mismatches and small insertion/deletion loops in DNA is performed by the MutS mismatch recognition protein. This highly conserved process exists in both prokaryotes and eukaryotes and failure to recognize DNA damage can have a detrimental effect on the fidelity of the genome and, in humans, has been linked to numerous forms of cancer. Several crystal structures have emerged over the years capturing MutS bound to various mismatches. However, the events directly following mismatch recognition remain unclear. To shed light on this matter, we present nine sub-μs, all-atom molecular dynamics simulations of Escherichia coli MutS bound to a G•T mismatch with different nucleotide configurations. From these simulations, we identified significant instability in the adjacent base 5′ to the thymine mismatch. In one case, the 5′ adjacent base completely loses base stacking and flips out spontaneously via the minor groove. To the best of our knowledge, this rare event is the first ever documented case of DNA base flipping from any unrestrained protein-DNA simulation. In addition, these observations are in excellent agreement with a recent experimental study where it was suggested that the 5′ adjacent base could exist in an extrahelical state. To further understand the energetics of base flipping in MutS, we utilized the Hamiltonian replica-exchange molecular dynamics (HREMD) method simulating 44 independent replicas in an explicit water box. The free energy profile generated from HREMD shows two distinct minima, one for the stacked state and one for flipped out state, separated by a small energy barrier with the flipped out state being the more favorable of the two. Together, our results offer an unprecedented level of new insight into the mismatch recognition process and further our knowledge of this complex system.