PMID:17630776

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Citation

Malta, E, Moolenaar, GF and Goosen, N (2007) Dynamics of the UvrABC nucleotide excision repair proteins analyzed by fluorescence resonance energy transfer.Biochemistry 46:9080-8

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.

Links

PubMed Online version:10.1021/bi7002235

Keywords

Adenosine Triphosphatases; Adenosine Triphosphate; Amino Acid Sequence; Amino Acid Substitution; Bacterial Proteins; Base Sequence; DNA; DNA Damage; DNA Helicases; DNA Repair; DNA-Binding Proteins; Dimerization; Endodeoxyribonucleases; Escherichia coli Proteins; Fluorescence Resonance Energy Transfer; Gene Deletion; Green Fluorescent Proteins; Luminescent Proteins; Models, Biological; Molecular Sequence Data; Protein Binding; Recombinant Fusion Proteins; Ultraviolet Rays

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Materials and Methods Used

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Phenotype Annotations

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Phenotype of Taxon Information Genotype Information (if known) Condition Information OMP ID OMP Term Name ECO ID ECO Term Name Notes Status

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain:
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: uvrAB
  • Genotype of Experimental Strain : uvrAB K45A
  • Reference Condition:

OMP:0000298

enzyme activity phenotype

ECO:0000049

reporter gene assay

ATP hydrolysis of uvrB by ATPase is inhibited (but not of uvrA in the complex). Table 1.


Notes

References

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