Signaling human mismatch repair by the formation of a hMSH2 -hMSH6 sliding DNA clamp

Scott David Gradia, Thomas Jefferson University


Germline mutations in several of the human DNA mismatch repair genes, including hMSH2, hMLH1, and hMSH6, are associated with Hereditary Non-Polyposis Colon Cancer. The loss of an intact DNA mismatch repair system and the development of a mutator phenotype is believed to result in carcinogenesis through the accumulation of secondary mutations. As mismatched DNA recognition complexes, the hMSH2-hMSH3 and hMSH2-hMSH6 heterodimers are responsible for initiating a DNA repair event by targeting the repair machinery to the site of the DNA mismatch. The hMSH2-hMSH6 heterodimer recognizes single base and small insertion/deletion DNA mismatches while hMSH2-hMSH3 recognizes small and large insertion/deletion DNA mismatches. This thesis provides a detailed analysis of the mismatched DNA binding and DNA stimulated ATPase activities of the hMSH2-hMSH6 heterodimeric protein. The finding that hMSH2-hMSH6 binds heteroduplex DNA in the ADP-bound form and releases it in the ATP-bound form supports the idea that this protein functions as a molecular switch. Recycling of hMSH2-hMSH6 mismatched DNA binding activity is accomplished by an intrinsic ATPase activity, which hydrolyzes ATP to ADP. In the absence of DNA, hMSH2-hMSH6 will hydrolyze a bound ATP and remain in an ADP bound form capable of binding mismatched DNA. Interaction with mismatched DNA stimulates hMSH2-hMSH6 to exchange ADP for ATP, and causes the ATP-bound protein to release the mismatched base pair by diffusing along the DNA backbone. If a free DNA end is available the ATP-bound hMSH2-hMSH6 will dissociate from the DNA lattice and hydrolyze its bound ATP, hence recycling mismatch-binding activity. Analysis of hMSH2-hMSH6 ATPase stimulation by various types of single base and insertion/deletion DNA mismatch substrates reveals a hierarchy of stimulation showing considerable similarity to previously reported mismatched DNA repair efficiencies in human cells. On the other hand, hMSH2-hMSH6 affinity for various DNA mismatches does not quantitatively agree with reported mismatch DNA repair efficiencies and reveals a strong bias for G/T mismatches. As a whole, this work supports a model for the initiation of bi-directional mismatch repair in which stochastic loading of multiple ATP-bound hMSH2-hMSH6 sliding clamps onto mismatch-containing DNA leads to recruitment of the repair machinery.

Subject Area

Molecular biology

Recommended Citation

Gradia, Scott David, "Signaling human mismatch repair by the formation of a hMSH2 -hMSH6 sliding DNA clamp" (1999). ProQuest ETD Collection - Thomas Jefferson University. AAI9963596.