UvrB domain 4, an autoinhibitory gate for regulation of DNA binding and ATPase activity.
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.
Wang
Hong
H
Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA.
DellaVecchia
Matthew J
MJ
Skorvaga
Milan
M
Croteau
Deborah L
DL
Erie
Dorothy A
DA
Van Houten
Bennett
B
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004249:DNA Damage; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000251:Adenosine Triphosphatases