Dynamics of the UvrABC Nucleotide Excision Repair Proteins Analyzed by Fluorescence Resonance Energy Transfer

Abstract

UvrB plays a key role in bacterial nucleotide excision repair. It is the ultimate damage-binding protein that interacts with both UvrA and UvrC. The oligomeric state of UvrB and the UvrAB complex have been subject of debate for a long time. Using fluorescence resonance energy transfer (FRET) between GFP and YFP fused to the C-terminal end of Escherichia coli UvrB, we unambiguously show that in solution two UvrB subunits bind to UvrA, most likely as part of a UvrA2B2 complex. This complex is most stable when both UvrA and UvrB are in the ATP-bound form. Analysis of a truncated form of UvrB shows that binding to UvrA promotes dimerization of the two C-terminal domain 4 regions of UvrB. The presence of undamaged DNA leads to dissociation of the UvrA2B2 complex, but when the ATPase site of UvrB is inactivated, the complex is trapped on the DNA. When the complex is bound to a damaged site, FRET between the two UvrB subunits could still be detected, but only as long as UvrA remains associated. Dissociation of UvrA from the damage-bound UvrB dimer leads to the reduction of the magnitude of the FRET signal, indicating that the domain 4 regions no longer interact. We propose that the UvrA-induced dimerization of the domain 4 regions serves to shield these domains from premature UvrC binding. Only after specific binding of the UvrB dimer to a damaged site and subsequent release of UvrA is the contact between the domain 4 regions broken, allowing recruitment of UvrC and subsequent incisions.

Single-molecule imaging of UvrA and UvrB recruitment to DNA lesions in living Escherichia coli

“…1a), and imaged using the same protocol as for UvrA. Because current models propose that NER is initiated by a stable complex of UvrA and UvrB3458910, and our data showed that UvrB is present at a similar level to UvrA (both ∼85 copies per cell; Fig. 3a and Supplementary Fig.…”

“…The observations that UvrA and UvrB are rarely complexed in solution in vivo , and that UvrA dimers can locate damage sites independently of UvrB, suggest that the current models, in which a stable UvrA–UvrB complex scans the genome to initiate NER34578910, need revision. We propose that NER initiation is a two-step process, where UvrA performs initial damage recognition and verification, and recruits UvrB from solution to perform further damage verification.…”

“…In vitro studies using purified proteins, including crystallographic studies of NER intermediates678, have provided a mechanistic framework of the initial steps in the NER pathway345678910. According to currently accepted models, dimeric UvrA forms a stable complex with either one or two UvrB molecules3451011.…”

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

“…Briefly, an UvrA•B heteromeric complex scans the DNA searching for damaged sites. The identity of the UvrA•B oligomeric state and of the searching complex have long been the subject of debate and controversy; however, two independent studies using fluorescence resonance energy transfer and single-molecule imaging of quantum-dot-labelled proteins have proposed that the ATP-bound UvrA 2 •B 2 complex is the stable form that slides along the DNA until a damaged site is encountered (8,9). Once recognized by the UvrA component, the injured strand is transferred to UvrB and UvrA is released.…”

UvrD Participation in Nucleotide Excision Repair Is Required for the Recovery of DNA Synthesis following UV-Induced Damage inEscherichia coli

“…Nucleotide excision repair is the process by which bulky adducts and lesions are removed and repaired from DNA [11, 12]. During nucleotide excision repair (NER), a heterotetramer, UvrA 2 UvrB 2 , recognizes and binds the damaged region [11, 13, 14]. UvrD acts after incision to release the resulting 10 to 12 bp oligonucleotide and UvrB-UvrC complex from the DNA [15–18].…”