Yue Zou

Strand opening by the UvrA2B complex allows dynamic recognition of DNA damage

Repair proteins alter the local DNA structure during nucleotide excision repair (NER). However, the precise role of DNA melting remains unknown. A series of DNA substrates containing a unique site‐specific BPDE‐guanine adduct in a region of non‐complementary bases were examined for incision by the Escherichia coli UvrBC endonuclease in the presence or absence of UvrA. UvrBC formed a pre‐incision intermediate with a DNA substrate containing a 6‐base bubble structure with 2 unpaired bases 5′ and 3 unpaired bases 3′ to the adduct. Formation of this bubble served as a dynamic recognition step in damage processing. UvrB or UvrBC may form one of three stable repair intermediates with DNA substrates, depending upon the state of the DNA surrounding the modified base. The dual incisions were strongly determined by the distance between the adduct and the double‐stranded–single‐stranded DNA junction of the bubble, and required homologous double‐stranded DNA at both incision sites. Remarkably, in the absence of UvrA, UvrBC nuclease can make both 3′ and 5′ incisions on substrates with bubbles of 3–6 nucleotides, and an uncoupled 5′ incision on bubbles of ≥≥10 nucleotides. These data support the hypothesis that the E.coli and human NER systems recognize and process DNA damage in a highly conserved manner.

FORMATION OF DNA REPAIR INTERMEDIATES AND INCISION BY THE ATP-DEPENDENT UVRB-UVRC ENDONUCLEASE

The Escherichia coli UvrB and UvrC proteins play key roles in DNA damage processing and incisions during nucleotide excision repair. To study the DNA structural requirements and protein-DNA intermediates formed during these processes, benzo[a]pyrene diol epoxide-damaged and structure-specific 50-base pair substrates were constructed. DNA fragments containing a preexisting 3′ incision were rapidly and efficiently incised 5′ to the adduct. Gel mobility shift assays indicated that this substrate supported UvrA dissociation from the UvrB-DNA complex, which led to efficient incision. Experiments with a DNA fragment containing an internal noncomplementary 11-base region surrounding the benzo[a]pyrene diol epoxide adduct indicated that UvrABC nuclease does not require fully duplexed DNA for binding and incision. In the absence of UvrA, UvrB (UvrC) bound to an 11-base noncomplementary region containing a 3′ nick (Y substrate), forming a stable protein-DNA complex (Kd ∼5-10 nM). Formation of this complex was absolutely dependent upon UvrC. Addition to this complex of ATP, but not adenosine 5′-(β,γ-iminotriphosphate) or adenosine 5′-(β,γ-methylene)triphosphate, caused incision three or four nucleotides 5′ to the double strand-single strand junction. The ATPase activity of native UvrB is activated upon interaction with UvrC and enhanced further by the addition of Y substrate. Incision of this Y structure occurs even without DNA damage. Thus the UvrBC complex is a structure-specific, ATP-dependent endonuclease.

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 coli proteins involved in removing DNA damage during the process of nucleotide excision repair. The relatively low concentrations (nanomolar) of the protein in the normal cells raise the potential questions about its stability in vivo under both normal and stress conditions. In vitro, UvrA at low concentrations is shown to be stabilized to heat inactivation by E. colimolecular chaperones DnaK or the combination of DnaK, DnaJ, and GrpE. These chaperone proteins allow sub-nanomolar concentrations of UvrA to load UvrB through >10 cycles of incision. Guanidine hydrochloride-denatured UvrA was reactivated by DnaK, DnaJ, and GrpE to as much as 50% of the native protein activity. Co-immunoprecipitation assays showed that DnaK bound denatured UvrA in the absence of ATP. UV survival studies of a DnaK-deficient strain indicated an 80-fold increased sensitivity to 100 J/m2 of ultraviolet light (254 nm) as compared with an isogenic wild-type 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 in nucleotide excision repair by maintaining repair proteins in their properly folded state.