Active site of (A)BC excinuclease. II. Binding, bending, and catalysis mutants of UvrB reveal a direct role in 3' and an indirect role in 5' incision.
UvrB plays a central role in (A)BC excinuclease. To study its role in the incision reactions, conserved His and Asp residues in this subunit were mutagenized. All His and the majority of Asp mutants behaved like wild-type protein in vivo and in vitro. However, three mutants, D337A, D478A, and D510A, either completely or partially abolished UvrB activity. All three mutant proteins associate with UvrA normally but D337A and D510A were unable to bind to DNA specifically. The UvrB-D478A mutant bound to DNA specifically but failed to denature and kink the DNA. However, UvrB-D478A was efficiently loaded onto DNA preincised at the 3' site and promoted near-normal incision by UvrC at the 5' site. We propose that D478 is involved in bending DNA and catalysis of the 3' incision and that the 3' incision precedes the 5' incision. UvrB which is missing the carboxyl-terminal 43 amino acids binds to, and kinks DNA but is unable to make the 3' incision suggesting that it is missing a residue involved in catalysis. This residue was identified to be E639 by site-specific mutagenesis.
Lin
J J
JJ
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599.
Phillips
A M
AM
Hearst
J E
JE
Sancar
A
A
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004265:DNA Helicases; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C058093:UvrB protein, E coli; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases