Abstract
The mechanics of hMSH2-hMSH6 ATP binding and hydrolysis are critical to several proposed mechanisms for mismatch repair (MMR), which in turn rely on the detailed coordination of ATP processing between the individual hMSH2 and hMSH6 subunits. Here we show that hMSH2-hMSH6 is strictly controlled by hMSH2 and magnesium in a complex with ADP (hMSH2(magnesium-ADP)-hMSH6). Destabilization of magnesium results in ADP release from hMSH2 that allows high affinity ATP binding by hMSH6, which then enhances ATP binding by hMSH2. Both subunits must be ATP-bound to efficiently form a stable hMSH2-hMSH6 hydrolysis-independent sliding clamp required for MMR. In the presence of magnesium, the ATP-bound sliding clamps remain on the DNA for ∼8 min. These results suggest a precise stepwise kinetic mechanism for hMSH2-hMSH6 functions that appears to mimic G protein switches, severely constrains models for MMR, and may partially explain the MSH2 allele frequency in Lynch syndrome or hereditary nonpolyposis colorectal cancer.
Highlights
The hMSH2-hMSH6 heterodimer must coordinate mismatch binding with dual site adenosine nucleotide processing
We find that hMSH2 in a complex with magnesium and adenosine nucleotide regulates all aspects of ADP/ATP processing of the hMSH2-hMSH6 heterodimer
We purified wild type hMSH2-hMSH6 (WT), hMSH2(K675A)-hMSH6 (2KA/ 6), hMSH2-hMSH6(K1140A) (2/6KA), and hMSH2(K675A)hMSH6(K1140A) (2KA/6KA) heterodimers and examined magnesium-dependent adenosine nucleotide cross-linking by the individual MutS homologs (MSH) subunits (Fig. 1, B and C) [12, 21, 31, 33]
Summary
The hMSH2-hMSH6 heterodimer must coordinate mismatch binding with dual site adenosine nucleotide processing. Destabilization of magnesium results in ADP release from hMSH2 that allows high affinity ATP binding by hMSH6, which enhances ATP binding by hMSH2 Both subunits must be ATP-bound to efficiently form a stable hMSH2-hMSH6 hydrolysis-independent sliding clamp required for MMR. In the presence of magnesium, the ATP-bound sliding clamps remain on the DNA for ϳ8 min These results suggest a precise stepwise kinetic mechanism for hMSH2-hMSH6 functions that appears to mimic G protein switches, severely constrains models for MMR, and may partially explain the MSH2 allele frequency in Lynch syndrome or hereditary nonpolyposis colorectal cancer. We find that hMSH2 in a complex with magnesium and adenosine nucleotide regulates all aspects of ADP/ATP processing of the hMSH2-hMSH6 heterodimer This central role of hMSH2 in the ATP processing may at least partially explain the allele frequency in Lynch syndrome or hereditary nonpolyposis colorectal cancer
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