Signaling human mismatch repair by the formation of a hMSH2-hMSH6 sliding DNA clamp
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. ^
Scott David Gradia,
"Signaling human mismatch repair by the formation of a hMSH2-hMSH6 sliding DNA clamp"
(January 1, 1999).
ETD Collection for Thomas Jefferson University.