Visualizing Spontaneous DNA Dynamics and its Role in Mismatch Recognition by Damage Recognition Protein Rad4

Abstract

The MutSg homologs, Msh4 and Msh5, play a significant role in meiotic recombination by assisting in the proper segregation of chromosomes through binding to DNA Holliday Junctions (HJ). We have employed fluorescent measurements with molecular dynamic simulations to generate a homologymodeled MD-refined structure of the protein-junction interaction. We have used this model coupled with our experimental results to examine Msh4-Msh5 conformational dynamics in DNA binding and identify the features of the Msh4-Msh5-junction interaction that lead to specific recognition. Our studies show that Msh4-Msh5 preferentially binds HJ DNA relative to duplex DNA and our model of the Msh4-Msh5-HJ interaction illustrates how Msh4-Msh5 stabilizes the stacked form of the junction. Stabilization of this junction conformation is generally refractory to branch migration, which is consistent with a potential role for Msh4-Msh5 (MutSg) in trapping HJ until they are resolved by Mlh1-Mlh3 (MutLg). Our homology modeled MD-refined structure of Msh4-Msh5 also revealed a putative DNA-binding region, in which the Msh4 subunit interacts more directly with either junction or duplex DNA relative to the Msh5 subunit. The model also indicates that the protein complex contains a second cavity, which could be important for binding other DNA substrates. Consequently, we have examined Msh4-Msh5 binding to six other recombination intermediates including DNA overhangs, forks, D-loop with single end strand invasion, pre-HJ and open junctions. Although nanomolar binding affinities are observed, the junction is the preferred substrate of Msh4-Msh5. Steady-state ATPase assays in the presence and absence of these different recombination intermediates elucidate the role of ATP hydrolysis in binding and specificity. The ability of the Msh4-Msh5 complex to specifically recognize these different recombination intermediates will be presented in terms of binding affinity, conformational dynamics, and utilization of ATP.

Impact of DNA sequences in the DNA duplex opening by the Rad4/XPC nucleotide excision repair complex

“…For instance, the more distorted and conformationally heterogeneous CCC/CCC mismatch had a greater specificity than a TAT/TAT mismatch that was structurally less distorted and heterogeneous. Furthermore, the more distorted sequences revealed more destabilization in thermal melting (51). However, it should be noted that thermal melting of a lesion-containing DNA duplex does not always correlate with NER.…”