The use of monoclonal antibodies for studying intermediates in DNA repair by the Escherichia coli Uvr(A)BC endonuclease.
The Escherichia coli Uvr(A)BC endonuclease acts in a progression of several distinct steps accompanied by changes in the conformation of macromolecular constituents, the overall architecture of the complex, and its stoichiometry. In order to probe these structural changes, we generated monoclonal antibodies (mAbs) to Uvr proteins. The anti-UvrA mAb, A2D1, recognizing the N-terminal zinc-finger region of UvrA, and the anti-UvrB mAb, B2E2, having an epitope within the 43 C-terminal amino acids of UvrB, were purified and further characterized. It was found that A2D1 mAb interacts in solution both with UvrA-UvrB and UvrA-DNA complexes in the presence of the requisite ATP. This implies that the N-terminal zinc-finger of UvrA doesn't play a direct role in its interactions with UvrB and DNA. On the other hand, A2D1 does inhibit formation of the UvrB-damaged DNA preincision complex, apparently by preventing UvrB delivery by UvrA. The interaction of B2E2 with UvrA-UvrB and nucleoprotein complexes, including UvrB, suggests that the highly hydrophobic C-terminal domain of UvrB (i) doesn't participate in its interaction with UvrA, (ii) is accessible to this mAb in an intermediate UvrA-UvrB DNA helix-tracking complex, and (iii) seems to be directly involved in the formation of the preincision complex. These conclusions are supported by the finding that the neutralizing effect of A2D1 and B2E2 on the Uvr(A)BC endonuclease is significantly decreased if the preincision complex is preformed prior to mAbs addition.
Kovalsky
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OI
Department of Biochemistry, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205.
Grossman
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D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000276:immunology; D004260:DNA Repair; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins / Q000276:immunology; D003852:Deoxyribonucleoproteins / Q000648:ultrastructure; D004706:Endodeoxyribonucleases / Q000276:immunology* / Q000378:metabolism; D018604:Epitope Mapping; D004926:Escherichia coli / Q000201:enzymology* / Q000276:immunology; D029968:Escherichia coli Proteins
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D029968:Escherichia coli Proteins; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC