Single-Molecule FRET to Measure Conformational Dynamics of DNA Mismatch Repair Proteins.

Single-molecule FRET measurements have a unique sensitivity to protein conformational dynamics. The FRET signals can either be interpreted quantitatively to provide estimates of absolute distance in a molecule configuration or can be qualitatively interpreted as distinct states, from which quantitative kinetic schemes for conformational transitions can be deduced. Here we describe methods utilizing single-molecule FRET to reveal the conformational dynamics of the proteins responsible for DNA mismatch repair.

MutL traps MutS at a DNA mismatch.

DNA mismatch repair (MMR) identifies and corrects errors made during replication. In all organisms except those expressing MutH, interactions between a DNA mismatch, MutS, MutL, and the replication processivity factor (β-clamp or PCNA) activate the latent MutL endonuclease to nick the error-containing daughter strand. This nick provides an entry point for downstream repair proteins.

A specific synergistic effect in ionic liquid chelate extraction based on neutral ternary chloro-complex formation.

In the chelate extraction of zinc(II) into an ionic liquid (IL), 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide (C4mimTf2N), with 8-(p-toluenesulfonamido)quinoline (Htsq), the co-existence of a certain amount of chloride anion in the aqueous phase resulted in an enhancement of the extractability. This enhancement did not occur at all in that into chloroform, a conventional organic extraction solvent. The specific synergistic effect was based on the dominant extraction of a neutral ternary complex, Zn(tsq)Cl, which is unstable in molecular organic solvents.

Biochemical analysis of the human mismatch repair proteins hMutSα MSH2(G674A)-MSH6 and MSH2-MSH6(T1219D).

The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2(G674A) or Msh6(T1217D) mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have a reduced lifespan due to loss of the MMR pathway (Lin, D. P.