| Popis: |
In E. coli the error rate of replication (10-6) is lowered another thousand-fold by the MutS, MutL and MutH system of proteins responsible for directing repair of base insertions, deletions, and mismatches. We provide here a study using magnetic trapping of single DNA molecules to analyze the interactions between these proteins and DNA containing a single indel mutation. MutS, albeit at relatively high concentrations, can be observed to stabilize small thermal loops of DNA on an indel-containing substrate, and this in an ATP-dependent manner; addition of MutL reduces the concentration of MutS required for looping to be observed. Loop size distributes roughly according to a J-factor distribution, and decreases as force increases, consistent with thermal loop stabilization by MutSL complexes. The reaction is allowed to proceed to the stage of DNA incision by the addition of MutH. With the further addition of T4 DNA ligase the steady-state rate of incision can be studied as a function of different parameters including DNA supercoiling, extending force, length of substrate DNA, or relative positioning between indel and incision sites. We compare these results to biophysical models for the protein-DNA interactions involved as well as stochastic simulations of the incision reaction occurring on linear DNA with a centrally-located indel and two symmetric proximal incision sites. Experimental results indicate that DNA incision can occur even in the absence of looping, and comparison to simulations supports the view that communication from DNA mismatch to DNA incision site takes place via thermal diffusion. |
