Selection of monoclonal antibodies for probing of functional intermediates in incision of UV-irradiated DNA by Uvr(A)BC endonuclease from Escherichia coli.
Monoclonal antibodies (mAbs) were generated that recognize UvrA and UvrB proteins. These proteins are components of the Uvr(A)BC endonuclease, which initiates nucleotide excision repair in Escherichia coli. mAbs, which can be used for probing of structural intermediates of Uvr(A)BC endonuclease functioning, were selected for their ability to: (i) recognize different epitopes; (ii) have a high-affinity for native antigenic protein; (iii) preserve functionality of the Uvr protein in immunocomplex. The adherence of anti-Uvr mAbs with these criteria was verified by additivity and competition tests, and by their influence on the ATPase activities of UvrA and UvrB*, the functionally active proteolytic fragment of UvrB. Two out of twelve anti-UvrA and seven out of thirteen anti-UvrB/anti-UvrB* hybridoma lines were shown to satisfy these criteria. Recognition of UvrA and UvrB deletion mutant proteins by mAbs was used to map their epitopes. Epitopes of A2D1 and A2B1 mAbs were mapped to regions of amino acids 230-281 and 560-680 of UvrA, respectively. Epitopes of anti-UvrB/UvrB* mAbs were assigned to the following amino acid regions of UvrB: B2A1, 8-61; B2C5 and B*2E3, 171-278; B2E2, 631-673; B3C1, 1-7 and/or 62-170; B*2B9, 473-630; B*3E11, 379-472. The ability of selected mAbs to neutralize the incision function of Uvr(A)BC was analyzed. The results are discussed in terms of the applicability of these mAbs to probe the structures of intermediates in the functioning of Uvr(A)BC.
Kovalsky
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OI
Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
Lin
C G
CG
Grossman
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D000251:Adenosine Triphosphatases / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001426:Bacterial Proteins / Q000276:immunology* / Q000378:metabolism; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism / Q000528:radiation effects*; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000276:immunology* / Q000378:metabolism; D004706:Endodeoxyribonucleases; D000939:Epitopes; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D014466:Ultraviolet Rays
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D000939:Epitopes; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; D004247:DNA; D004706:Endodeoxyribonucleases; C066567:UvrC protein, E coli; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases