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The entire process of nucleotide excision repair (NER) in Escherichia coli has been reconstituted in vitro from purified proteins and defined DNA substrates. However, how this system is organized in vivo in unclear. We report here the isolation and characterization of macromolecular assemblies containing NER and transcription proteins from E. coli. This ensemble consists of at least 17 proteins. They are recruited, as a consequence of DNA damage induced by UV irradiation, to the inner membrane. The UV-induced 6-4 photoproducts are also relocated to the inner membrane following UV-irradiation of the cells. This recruitment process is dependent on the uvrA, uvrC and recA gene products. These results suggest that at least part of the repair process may associate with the inner membrane and also provide insights into understanding the cellular organization of repair processes.

A newly developed cell-free system was used to study DNA repair synthesis carried out by extracts from human cell lines in vitro. Extracts from a normal human lymphoid cell line and from cell lines established from individuals with hereditary dysplastic nevus syndrome perform damage-dependent repair synthesis in plasmid DNA treated with cis- or trans-diamminedichloro-platinum(II) or irradiated with ultraviolet light. Cell extracts of xeroderma pigmentosum origin (complementation groups A, C, D, and G) are deficient in DNA repair synthesis. When damaged plasmid DNA was pretreated with purified Escherichia coli UvrABC proteins, xeroderma pigmentosum cell extracts were able to carry out DNA repair synthesis. The ability of E. coli UvrABC proteins to complement xeroderma pigmentosum cell extracts indicates that the extracts are deficient in incision, but can carry out later steps of repair. Thus the in vitro system provides results that are in agreement with the incision defect found from studies of xeroderma pigmentosum cells.

We have analyzed the ATPase activity exhibited by the UvrABC DNA repair complex. The UvrA protein is an ATPase whose lack of DNA dependence may be related to the ATP induced monomer-dimer transitions. ATP induced dimerization may be responsible for the enhanced DNA binding activity observed in the presence of ATP. Although the UvrA ATPase is not stimulated by dsDNA, such DNA can modulate the UvrA ATPase activity by decreases in Km and Vm and alterations in the Ki for ADP and ATP-gamma-S. The induction of such changes upon binding to DNA may be necessary for cooperative interactions of UvrA with UvrB that result in a DNA stimulated ATPase for the UvrAB protein complex. The UvrAB ATPase displays unique kinetic profiles that are dependent on the structure of the DNA effector. These kinetic changes correlate with changes in footprinting patterns, the stabilization of protein complexes on DNA damage and with the expression of helicase activity.

UvrB is specifically proteolyzed in Escherichia coli cell extracts to UvrB*. UvrB* is capable of interacting with UvrA in an apparently similar manner to the UvrB, however UvrB* is defective in the DNA strand displacement activity normally displayed by UvrAB. Whereas the binding of UvrC to a UvrAB-DNA complex leads to DNA incision and persistence of a stable post-incision protein-DNA complex, the binding of UvrC to UvrAB* leads to dissociation of the protein complex and no DNA incision is seen. The factor which stimulates this proteolysis has been partially purified and its substrate specificity has been examined. The protease factor is induced by "stress" and is under control of the htpR gene. The potential role of this proteolysis in the regulation of levels of active repair enzymes in the cell is discussed.

The incision of damaged DNA by the Escherichia coli UvrABC endonuclease requires ATP hydrolysis. Although the deduced sequence of the UvrB protein suggests a putative ATP binding site, no nucleoside triphosphatase activity is demonstrable with the purified UvrB protein. The UvrB protein is specifically proteolyzed in E. coli cell extracts to yield a 70 kD fragment, referred to as UvrB*, which has been purified and is shown to possess a single-strand DNA dependent ATPase activity. Substrate specificity and kinetic analyses of UvrB* catalyzed nucleotide hydrolysis indicate that the stimulation in DNA dependent ATPase activity following formation of the UvrAB complex results from the activation of the normally sequestered UvrB associated ATPase. Using nucleotide analogues, it can be shown that this activity is essential to the DNA incision reaction carried out by the UvrABC complex.

The effects of the binding of the E. coli UvrA and UvrB proteins on the linking number (delta L) of superhelical DNA has been measured. The effects of cofactor ATP structure on UvrAB-nucleoprotein complex formation revealed that nucleotide binding, not hydrolysis, is sufficient to locally unwind the DNA helix of both ultraviolet light-damaged as well as undamaged DNAs. The extent of this unwinding is of the same order of magnitude as the nucleotide distances of the double incision sites generated by the UvrABC endonucleolytic reaction.

Incision of damaged DNA by the Escherichia coli UvrABC endonuclease requires the UvrA, UvrB, and UvrC proteins as well as ATP hydrolysis. This incision reaction can be divided into three steps: site recognition, preincision complex formation, and incision. UvrAB is able to execute the first two steps in the reaction while the addition of UvrC is required for the incision of DNA. This incision reaction does not require ATP hydrolysis and results in the formation of a tight UvrABC post-incision complex and the generation of an oligomer of approximately 12 nucleotides. At high UvrABC concentrations the specificity of the incision for damaged DNA is decreased and significant incision of undamaged DNA occurs. Analogous to damage specific incision, this type of incision leads to generation of an oligonucleotide, but in this case the size is approximately 9 nucleotides in length. Further evidence shows that the combination of UvrB and UvrC proteins can generate a significant amount of a similar size product on undamaged DNA. In addition, the UvrC protein alone can generate a small amount of the same product. Immunological characterization of the weak nuclease activity seen with UvrC indicates that the activity is very tightly associated with the purified UvrCmore » protein.« less

The UvrA, UvrB and UvrC proteins of Escherichia coli have been purified in good yields to homogeneity with rapid three- or four-step purification procedures. The cloned uvrA and uvrB genes were placed under control of the E. coli bacteriophage lambda PL promoter for amplification of expression. Expression of the uvrC gene could not be amplified by this strategy, however, subcloning of this gene into the replication-defective plasmid pRLM24 led to significant overproduction of the UvrC protein. The purified UvrA protein, with its associated ATPase activity, has a molecular weight of 114,000, the purified UvrB is an 84,000 molecular weight protein and the UvrC protein has a molecular weight of 67,000.

An examination has been made into the nature of the nucleoprotein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease when acting on a pyrimidine dimer-containing fd RF-I DNA species. The complexes of proteins and DNA form in unique stages. The first stage of binding involves an ATP-stimulated interaction of the UvrA protein with duplex DNA containing pyrimidine dimer sites. The UvrB protein significantly stabilizes the UvrA-pyrimidine dimer containing DNA complex which, in turn, provides a foundation for the binding of UvrC to activate the UvrABC endonuclease. The binding of one molecule of UvrC to each UvrAB-damaged DNA complex is needed to catalyze incision in the vicinity of pyrimidine dimer sites. The UvrABC-DNA complex persists after the incision event suggesting that the lack of UvrABC turnover may be linked to other activities in the excision-repair pathway beyond the initial incision reaction.