INVOLVEMENT OF MOLECULAR CHAPERONINS IN NUCLEOTIDE EXCISION-REPAIR - DNAK LEADS TO INCREASED THERMAL-STABILITY OF UVRA, CATALYTIC UVRB LOADING, ENHANCED REPAIR, AND INCREASED UV RESISTANCE

UvrA is one of the key Escherichia coil proteins involved in removing DNA damage during the process of nucleotide excision repair. The relat ively low concentrations (nanomolar) of the protein in the normal cell s raise the potential questions about its stability in vivo under both normal and stress conditions. In, vitro, UvrA at low concentrations i s shown to be stabilized to heat inactivation by E. coil molecular cha perones DnaK or the combination of DnaK, DnaJ, and GrpE. These chapero ne proteins allow sub-nanomolar concentrations of UvrA to load UvrB th rough >10 cycles of incision. Guanidine hydrochloride-denatured UvrA w as reactivated by DnaK, DnaJ, and GrpE to as much as 50% of the native protein activity. Co-immunoprecipitation assays showed that DnaK boun d denatured UvrA in the absence of ATP. UV survival studies of a DnaK- deficient strain indicated an 80-fold increased sensitivity to 100 J/m (2) of ultraviolet light (254 nm) as compared with an isogenic wild-ty pe strain. Global repair analysis indicated a reduction in the extent of pyrimidine dimer and 6-4 photoproduct removal in the DnaK-deficient cells. These results suggest that molecular chaperonins participate i n nucleotide excision repair by maintaining repair proteins in their p roperly folded state.