The (A)BC excinuclease of Escherichia coli has only the UvrB and UvrC subunits in the incision complex.

Abstract

The uvrA, uvrB, and uvrC genes control excision repair in Escherichia coli. Cells with mutations in any of these three genes cannot repair DNA by nucleotide excision. When the purified gene products-the UvrA, UvrB, and UvrC proteins-are mixed together, an excision nuclease is formed that incises on both sides of the damaged nucleotide in an ATP-dependent reaction; it has been presumed that the excision nuclease was an ABC complex containing all three Uvr proteins. To determine the stoichiometry of the subunits in the enzyme, we conducted hydrodynamic studies with mixtures of the subunits with or without DNA substrate. We found that without DNA the UvrA subunit is a dimer and that when UvrB protein is also present, a (UvrA)2(UvrB), complex forms.Without DNA no detectable interaction of either the UvrA or UvrB subunits or the (UvrA)2(UvrB), complex with the UvrC subunit occurs. Unexpectedly, with UV-irradiated DNA, the UvrA/UvrB ratio in isolated DNA-protein complexes is variable, and the ratio becomes infinitesimally low as the UvrA concentration in the reaction mixture decreases. Under conditions of saturating UvrB protein approximately one UvrB molecule binds to DNA per damaged site in a reaction that requires catalytic amounts of UvrA subunit. Addition of UvrC protein to purified UvrB-DNA complexes results in rapid incision of the DNA, presumably catalyzed by an excision nuclease containing only UvrB and UvrC subunits.The UvrA, UvrB, and UvrC proteins, referred to collectively as "ABC excinuclease," initiate nucleotide excision repair in Escherichia coli by incising seven bases 5' and four bases 3' to nucleotide mono-and diadducts in DNA (1) We have conducted physical studies to determine the stoichiometry of the subunits in preincision complexes of the Uvr proteins in the absence and presence of DNA. We found that UvrA dimerizes and, in an ATP-dependent reaction, associates with UvrB to form a (UvrA)2(UvrB), complex in the absence of DNA. More importantly, we have determined that UvrA, possibly as the (UvrA)2(UvrB), complex, delivers UvrB onto damaged DNA and then dissociates from the UvrB-DNA complex. Upon addition of UvrC, the DNA in the UvrB-DNA complex is incised in a manner typical of the so-called ABC excinuclease. Thus, our data indicate that UvrA is essential for the formation of the nucleotide excision nuclease complex, but it is not involved in the incision reactions. MATERIALS AND METHODSEnzymes and Substrates. The UvrA, UvrB, and UvrC proteins were purified to >909% homogeneity as described (4). Phage T4 endonuclease V was purchased from Applied Genetics (Freeport, NY). Protein standards for hydrodynamic analyses were from Bio-Rad and M13mp19RF DNA was from Boehringer Mannheim. 3H-labeled pBR322 DNA (1.1 x 104 cpm/,ug) was purified from E. coli AB2487/ pBR322 by Sarkosyl lysis followed by two cesium chloride/ ethidium bromide density centrifugations. When indicated, DNA was damaged by UV irradiation (25-375 J/m2) by using a Sylvania germicidal lamp (254 nm). The number of pyrimidine dimers...

Slx1-Slx4 Are Subunits of a Structure-specific Endonuclease That Maintains Ribosomal DNA in Fission Yeast

“…15, January 2004also required for the 3Ј incision (Orren and Sancar, 1989;Verhoeven et al, 2000). UvrB is thought to stabilize the UvrC-DNA contacts.…”

The Nobel Prize in Chemistry 2015: Exciting discoveries in DNA repair by Aziz Sancar

“…Hydrodynamic studies that I performed in Aziz's lab showed that UvrB and UvrC in isolation were monomeric, while UvrA alone formed dimers (Orren and Sancar, 1989). Mixtures of the proteins in the absence of DNA revealed a (UvrA) 2 UvrB complex, and UvrC did not associate with this complex or with UvrA or UvrB alone (Orren and Sancar, 1989).…”

“…Hydrodynamic studies that I performed in Aziz's lab showed that UvrB and UvrC in isolation were monomeric, while UvrA alone formed dimers (Orren and Sancar, 1989). Mixtures of the proteins in the absence of DNA revealed a (UvrA) 2 UvrB complex, and UvrC did not associate with this complex or with UvrA or UvrB alone (Orren and Sancar, 1989). UvrA was shown to be instrumental in DNA damage recognition, with higher affinity to damaged than to undamaged DNA, a difference that was further stimulated by ATP; UvrA bound to a 33 bp region surrounding a psoralen adduct (Seeberg and Steinum, 1982;Van Houten et al, 1987;Yeung et al, 1986).…”

UvrA and UvrB suppress illegitimate recombination: Synergistic action with RecQ helicase

“…UvrA 2 -UvrB 1 complexes recognize DNA damage. Then, the UvrA proteins dissociate from the UvrB-DNA complex, and a UvrC protein binds to the UvrB-DNA complex (15), leading to incisions on either side of the damage. Finally, the UvrD helicase removes the damaged region.…”