Abstract

UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA. Regulation of DNA binding and ATP hydrolysis by UvrB is poorly understood. Using atomic force microscopy and biochemical assays, we found that truncation of domain 4 of Bacillus caldotenax UvrB (UvrBDelta4) leads to multiple changes in protein function. Protein dimerization decreases with an approximately 8-fold increase of the equilibrium dissociation constant and an increase in DNA binding. Loss of domain 4 causes the DNA binding mode of UvrB to change from dimer to monomer, and affinity increases with the apparent dissociation constants on nondamaged and damaged single-stranded DNA decreasing 22- and 14-fold, respectively. ATPase activity by UvrBDelta4 increases 14- and 9-fold with and without single-stranded DNA, respectively, and UvrBDelta4 supports UvrA-independent damage-specific incision by Cho on a bubble DNA substrate. We propose that other than its previously discovered role in regulating protein-protein interactions, domain 4 is an autoinhibitory domain regulating the DNA binding and ATPase activities of UvrB.

Highlights

  • UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA

  • Loss of domain 4 causes the DNA binding mode of UvrB to change from dimer to monomer, and affinity increases with the apparent dissociation constants on nondamaged and damaged single-stranded DNA decreasing 22- and 14-fold, respectively

  • It is believed that UvrA, as a dimer within the UvrAB complex, first recognizes helical distortions induced by DNA damage

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Summary

Introduction

UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA. We show that truncation of domain 4 (residues Pro612 to Gly658) of UvrB leads to: 1) a change in UvrB DNA binding mode and increase in DNA binding affinity; 2) an increase in UvrB ATPase activity; and 3) UvrA-independent, damagespecific incision of a bubble substrate mediated by Cho (UvrC homolog). These data suggest that DNA repair enzymes may represent another important class of DNA-binding proteins that are regulated by autoinhibitory domains

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