Exploring the Interaction of Msh2‐Msh6 with the Holliday Junction Through ATPase Activity

Abstract

DNA mismatch repair systems (MMR) are important in reducing post replicative errors and preventing recombination between homologous sequences. Mismatch repair is highly conserved in most organisms and is initiated by the MutS homodimer in prokaryotes. In eukaryotes, small insertion/deletion loops (IDL), and single base pair mismatches are recognized by the MutS homolog Msh2‐Msh6. Prior research in our lab and by others (Marsischky G. T., Lee S., Griffith J., and Kolodner R. D. (1999) J.Biol.Chem. 274: 7200–7206 doi:10.1074/jbc.274.11.7200) determined the binding affinity of the junction with Msh2‐Msh6 with a Kd = 12.9 ± 1.3 nM, which is similar to the binding affinity for a G:T duplex, suggesting that the Msh2‐Msh6 Holliday Junction interaction is specific. Our investigation aims to understand the Msh2‐Msh6‐junction binding interaction by studying nucleotide utilization upon binding and determining any conformational changes associated with complex formation and ATP hydrolysis. We use the well‐characterized J3 Holliday junction with 17 base pair arms to characterize binding. Malachite green assays measuring inorganic phosphate were performed to obtain the rate of ATP hydrolysis in the presence of Msh2‐Msh6, J3 junction, and other DNA substrates. Msh2‐Msh6 alone has a Kcat = 0.27s−1 in the presence of 100mM NaCl, with the Kcat weakly increasing with a G:T mismatch 34‐mer duplex and the J3 junction to 0.47s−1and 0.6s−1 respectively. We will also report on junction conformational changes associated with protein binding and how protein affinity for the junction is modulated by nucleotide binding.Support or Funding InformationASBMB Undergraduate Research Grant.