Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
Binding Dynamics of Yeast MutS Homologs Msh4-Msh5 with the Holliday Junction
MutS homologs (Msh) are conserved in all organisms and they are involved in the process of mismatch recognition and repair. In Saccharomyces cerevisiae there are five MutS Homologs (Msh2 through Msh6) that participate in repair and recombination processes. The heterodimeric protein complexes, Msh2-Msh3 and Msh2-Msh6, play a role in mismatch repair. MutS homolog Msh4 and Msh5 are not involved in mismatch recognition and repair and genetic studies in S. cerevisiae and mouse indicate that Msh4-Msh5 are responsible for gamete viability during meiosis, recombination and chromosomal segregation. Recent studies have shown that human Msh4-Msh5 recognizes and stabilizes Holliday or four-way DNA Junctions (4WJ), an intermediate in double stranded break repair. Our efforts are focused on elucidating structure-function relationships of S. cerevisiae MutS homolog Msh4-Msh5 by specifically studying the binding interaction of Msh4-Msh5 with DNA 4WJs. We have successfully expressed and purified S. cerevisiae Msh4-Msh5 in E. coli cells as both a heterodimeric protein complex as well as Msh4 and Msh5 monomers. Our initial data demonstrates that the purified proteins are active and binds with high affinity to DNA 4WJ. The binding affinity is in the sub nanomolar range and lower than that of human Msh4-Msh5 reported previously. To study the effect of Msh4-Msh5 on junction conformation, we are using Förster resonance energy transfer. These measurements also probe whether Msh4-Msh5 preferentially binds to the open or stacked form of the junction. These measurements will also address whether stabilization of the stacked conformation is functionally related to suppression of recombination.
This research has been supported by NSF Grant MCB-0316625, MCB-0843656.
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Nishant, K. T., Chen, C., Shinohara, M., Shinohara, A., & Alani, E. (2010). Genetic analysis of baker's yeast Msh4-Msh5 reveals a threshold crossover level for meiotic viability. PLoS Genetics, 6(8). doi:10.1371/journal.pgen.1001083.
Department of Molecular Biology and Biochemistry