title: Single-molecule analysis reveals two separate DNA-binding domains in the Escherichia coli UvrA dimer.
abstract: The UvrA protein is the initial damage-recognizing factor in bacterial nucleotide excision repair. Each monomer of the UvrA dimer contains two ATPase sites. Using single-molecule analysis we show that dimerization of UvrA in the presence of ATP is significantly higher than with ADP or nonhydrolyzable ATPgammaS, suggesting that the active UvrA dimer contains a mixture of ADP and ATP. We also show that the UvrA dimer has a high preference of binding the end of a linear DNA fragment, independent on the presence or type of cofactor. Apparently ATP binding or hydrolysis is not needed to discriminate between DNA ends and internal sites. A significant number of complexes could be detected where one UvrA dimer bridges two DNA ends implying the presence of two separate DNA-binding domains, most likely present in each monomer. On DNA containing a site-specific lesion the damage-specific binding is much higher than DNA-end binding, but only in the absence of cofactor or with ATP. With ATPgammaS no discrimination between a DNA end and a DNA damage could be observed. We present a model where damage recognition of UvrA depends on the ability of both UvrA monomers to interact with the DNA flanking the lesion.
authors.affiliation: Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands.
authors.forename: Koen
authors.initials: K
authors.lastname: Wagner
authors.forename: Geri
authors.initials: G
authors.lastname: Moolenaar
authors.forename: John
authors.initials: J
authors.lastname: van Noort
authors.forename: Nora
authors.initials: N
authors.lastname: Goosen
mesh_terms: D000244:Adenosine Diphosphate / Q000032:analysis; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases