Potential role of proteolysis in the control of UvrABC incision.

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.     

Involvement of a cryptic ATPase activity of UvrB and its proteolysis product, UvrB* in DNA repair

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.     

Incision of damaged versus nondamaged DNA by the Escherichia coli UvrABC proteins.

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 UvrC protein.     

Analysis of UvrABC endonuclease reaction intermediates on cisplatin-damaged DNA using mobility shift gel electrophoresis.

One of the least understood steps in the UvrABC mediated excision repair process is the recognition of lesions in the DNA. The isolation of different reaction intermediates is of vital importance for the unraveling of the mechanism. A mobility shift gel electrophoresis assay is described which visualizes such intermediates. After incubation of a DNA substrate containing a specific cisplatin adduct with UvrA alone or with UvrA and UvrB, UvrA.DNA, UvrAB.DNA and UvrB.DNA complexes were observed which could be identified using specific antibodies. At low UvrA concentrations in the presence of UvrB only the UvrB.DNA complex is observed. Bands corresponding to the UvrAB.DNA complex and also other nonspecific bands are found at relatively high UvrA concentrations. The DNase-I footprint for the UvrAB.- and UvrB.DNA complex are very similar and protect about 20 bases. Both complexes are incised in the presence of UvrC with comparable efficiency. The UvrAB.- and the UvrB.DNA complex were both incised at the 8th phosphodiester bond 5' to a specific cisplatin adduct. In addition the UvrAB.DNA complex could also be incised at the 15th phosphodiesterbond 5' to the damaged site. The results suggest that the UvrB.DNA complex is the natural substrate for UvrC-induced incision.     

Repair of DNA-containing pyrimidine dimers

Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.     

Dynamics of the UvrABC nucleotide excision repair proteins analyzed by fluorescence resonance energy transfer

UvrB plays a key role in bacterial nucleotide excision repair. It is the ultimate damage-binding protein that interacts with both UvrA and UvrC. The oligomeric state of UvrB and the UvrAB complex have been subject of debate for a long time. Using fluorescence resonance energy transfer (FRET) between GFP and YFP fused to the C-terminal end of Escherichia coli UvrB, we unambiguously show that in solution two UvrB subunits bind to UvrA, most likely as part of a UvrA2B2 complex. This complex is most stable when both UvrA and UvrB are in the ATP-bound form. Analysis of a truncated form of UvrB shows that binding to UvrA promotes dimerization of the two C-terminal domain 4 regions of UvrB. The presence of undamaged DNA leads to dissociation of the UvrA2B2 complex, but when the ATPase site of UvrB is inactivated, the complex is trapped on the DNA. When the complex is bound to a damaged site, FRET between the two UvrB subunits could still be detected, but only as long as UvrA remains associated. Dissociation of UvrA from the damage-bound UvrB dimer leads to the reduction of the magnitude of the FRET signal, indicating that the domain 4 regions no longer interact. We propose that the UvrA-induced dimerization of the domain 4 regions serves to shield these domains from premature UvrC binding. Only after specific binding of the UvrB dimer to a damaged site and subsequent release of UvrA is the contact between the domain 4 regions broken, allowing recruitment of UvrC and subsequent incisions.     

Involvement of a cryptic ATPase activity of UvrB and its proteolysis product, UvrB* in DNA repair.

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.     

Post-incision steps of nucleotide excision repair in Escherichia coli. Disassembly of the UvrBC-DNA complex by helicase II and DNA polymerase I.

UvrA, UvrB, and UvrC initiate nucleotide excision repair by incising a damaged DNA strand on each side of the damaged nucleotide. This incision reaction is substoichiometric with regard to UvrB and UvrC, suggesting that both proteins remain bound following incision and do not "turn over." The addition of only helicase II to such reaction mixtures turns over UvrC; UvrB turnover requires the addition of helicase II, DNA polymerase I, and deoxynucleoside triphosphates. Column chromatography and psoralen photocross-linking experiments show that following incision, the damaged oligomer remains associated with the undamaged strand, UvrB, and UvrC in a post-incision complex. Helicase II releases the damaged oligomer and UvrC from this complex, making repair synthesis possible; DNase I footprinting experiments show that UvrB remains bound to the resulting gapped DNA until displaced by DNA polymerase I. The specific binding of UvrB to a psoralen adduct in DNA inhibits psoralen-mediated DNA-DNA cross-linking, yet promotes the formation of UrvB-psoralen-DNA cross-links. The discovery of psoralen-UvrB photocross-linking offers the potential of active-site labeling.     

Robust incision of Benoz[a]pyrene-7,8-dihyrodiol-9,10-epoxide-DNA adducts by a recombinant thermoresistant interspecies combination UvrABC endonuclease system

Prokaryotic DNA repair nucleases are useful reagents for detecting DNA lesions. UvrABC endonuclease, encoded by the UvrA, UvrB, and UvrC genes can incise DNA containing bulky nucleotide adducts and intrastrand cross-links. UvrA, UvrB, and UvrC were cloned from Bacillus caldotenax (Bca)and UvrC from Thermatoga maritima (Tma), and recombinant proteins were overexpressed in and purified from Escherichia coli. Incision activities of UvrABC composed of all Bca-derived subunits (UvrABC(Bca)) and an interspecies combination UvrABC composed of Bca-derived UvrA and UvrB and Tma-derived UvrC (UvrABC(Tma)) were compared on benoz[a]pyrene-7,8-dihyrodiol-9,10-epoxide (BPDE)-adducted substrates. Both UvrABC(Bca) and UvrABC(Tma) specifically incised both BPDE-adducted plasmid DNAs and site-specifically modified 50-bp oligonucleotides containing a single (+)-trans- or (+)-cis-BPDE adduct. Incision activity was maximal at 55-60 degrees C. However, UvrABC(Tma) was more robust than UvrABC(Bca) with 4-fold greater incision activity on BPDE-adducted oligonucleotides and 1.5-fold greater on [(3)H]BPDE-adducted plasmid DNAs. Remarkably, UvrABC(Bca) incised only at the eighth phosphodiester bond 5' to the BPDE-modified guanosine. In contrast, UvrABC(Tma) performed dual incision, cutting at both the fifth phosphodiester bond 3' and eighth phosphodiester bond 5' from BPDE-modified guanosine. BPDE adduct stereochemistry influenced incision activity, and cis adducts on oligonucleotide substrates were incised more efficiently than trans adducts by both UvrABC(Bca) and UvrABC(Tma). UvrAB-DNA complex formation was similar with (+)-trans- and (+)-cis-BPDE-adducted substrates, suggesting that UvrAB binds both adducts equally and that adduct configuration modifies UvrC recognition of the UvrAB-DNA complex. The dual incision capabilities and higher incision activity of UvrABC(Tma) make it a robust tool for DNA adduct studies.     

A Physical Interaction of UvrD with Nucleotide Excision Repair Protein UvrB

The dual-incision nature of the reaction of UV-irradiated DNA catalyzed by the UvrABC complex potentially leads to excision of a damaged fragment. However, neither fragment release under nondenaturing conditions nor the UvrBC proteins are turned over. The addition of the UvrD protein to the incised DNA-UvrBC complex results in excision of the incised damaged strand and in the turnover of the UvrC protein. In an effort to better understand the involvement of UvrD in the excision step, immunoprecipitation was used to detect interacting proteins with UvrD in the DNA repair. In this communication, it is shown that UvrA and UvrB are precipitated with UvrD in solution but the UvrAB complex is not. In the incision complex, UvrB could be precipitated and the preincubation of UvrD with UvrB revealed an inhibitory effect on the turnover of the incision complex. These data imply that UvrB in the incision complex seems to recruit UvrD to the 3 incised site of the incised strand by protein-protein interaction and to allow initiation of unwinding by UvrD from the resulting nick in a 3 to 5 direction.     

Interaction of the UvrABC endonuclease with DNA containing a psoralen monoadduct or cross-link. Differential effects of superhelical density and comparison of preincision complexes.

The effect of negative supercoiling on UvrABC incision of covalently closed duplex DNA circles containing either a furan-side monoadduct or a cross-link of 4'-hydroxymethyl-4,5',8-trimethylpsoralen at a unique site was examined. The rate of UvrABC incision of these DNA substrates was measured as a function of superhelical density, sigma, for values of sigma between 0 and -0.050. The monoadducted DNA substrate was incised at close to the maximum rate at all superhelical densities, with only a slight stimulation of activity between sigma = 0 and -0.035. In contrast, efficient UvrABC incision of the cross-linked DNA substrate required the DNA to be underwound, and activity showed a linear dependence on superhelical density up to sigma = -0.035. DNase I protection studies show that in the presence of both UvrA and UvrB a protein complex binds to the site of a psoralen monoadduct or cross-link in linear DNA. This UvrA-UvrB-dependent complex binds with similar affinity to both the monoadducted and the cross-linked DNA helices. However, differences in the DNase I footprint on these two DNA substrates indicate that the interaction of this protein complex is different at these two lesions. The addition of UvrC to linear DNA molecules that are saturated at the site of the lesion with the UvrA-UvrB-dependent complex resulted in efficient nicking of the monoadducted DNA, but not the cross-linked DNA. Thus, the properties of a DNA lesion site that lead to UvrAB recognition and binding are not necessarily sufficient to allow incision when all three Uvr subunits are present. We propose that after recognition and binding of a lesion site by the UvrAB complex and prior to incision, the damaged DNA helix undergoes a conformational change such as unwinding or melting that is induced by the lesion-bound Uvr complex.     

A Structural Model for the Damage-sensing Complex in Bacterial Nucleotide Excision Repair

Nucleotide excision repair is distinguished from other DNA repair pathways by its ability to process a wide range of structurally unrelated DNA lesions. In bacteria, damage recognition is achieved by the UvrA·UvrB ensemble. Here, we report the structure of the complex between the interaction domains of UvrA and UvrB. These domains are necessary and sufficient for full-length UvrA and UvrB to associate and thereby form the DNA damage-sensing complex of bacterial nucleotide excision repair. The crystal structure and accompanying biochemical analyses suggest a model for the complete damage-sensing complex.Nucleotide excision repair is distinguished from other DNA repair pathways by its ability to process a diverse set of lesions. In bacteria, the initial steps are carried out by three proteins: UvrA, UvrB, and UvrC. The UvrA·UvrB complex conducts surveillance of DNA and recognizes damage. Having located a lesion, UvrA “loads” UvrB onto the DNA at the damaged sites and then dissociates. Damage searching, formation of the UvrB·DNA “preincision” complex, and dissociation of UvrA are regulated by ATP (1). UvrB subsequently recruits the endonuclease UvrC, which catalyzes incisions on either side of the lesion (2, 3). Following incision, UvrC and the damage-containing oligonucleotide are removed by UvrD (helicase II), whereas UvrB remains bound to the gapped DNA and recruits DNA polymerase I for repair synthesis. Sealing of the single-stranded nick completes the repair process and restores the original DNA sequence (4).Since its discovery more than 40 years ago, bacterial nucleotide excision repair has been extensively studied, resulting in a large body of work that describes the protein components and the details of how they operate. Notwithstanding the trove of genetic and biochemical data, several key questions remain unanswered. For example, how does the same set of proteins handle a diverse set of lesions while maintaining specificity? How do UvrA and UvrB cooperate during damage recognition, and what is the precise role of ATP? Ongoing studies in the field, including those described below, aim to address these issues.Recently, we reported the structure of Geobacillus stearothermophilus UvrA and the identification of binding sites for DNA and UvrB (5). We also established that the identified UvrB-binding domain is necessary and sufficient to mediate the UvrA-UvrB interaction and that the isolated interaction domains of UvrA (5) and UvrB (6) bind to each other in solution.To understand the interaction between UvrA and UvrB, we have determined the crystal structure of the complex between the two isolated interaction domains. The structure revealed that UvrA-UvrB interaction interface is largely polar, mediated by several highly conserved charged residues. Site-directed mutagenesis and biochemical characterization of the mutant proteins confirmed the importance of the observed interactions. Based on the interaction domain complex structure, we have constructed a structural model for the full-length UvrA·UvrB ensemble and propose two models for lesion recognition that will serve as a basis for future experiments.     

The C-terminal region of the Escherichia coli UvrC protein, which is homologous to the C-terminal region of the human ERCC1 protein, is involved in DNA binding and 5'-incision.

The incisions in the DNA at the 3'- and 5'-side of a DNA damage during nucleotide excision repair in Escherichia coli occur in a complex consisting of damaged DNA, UvrB and UvrC. The exact requirements for the two incision events, however, are different. It has previously been shown that the 3'-incision requires the interaction between the C-terminal domain of UvrB and a homologous region in UvrC. This interaction, however, is dispensable for the 5'-incision. Here we show that the C-terminal domain of the UvrC protein is essential for the 5'-incision, whereas this domain can be deleted without affecting the 3'-incision. The C-terminal domain of UvrC is homologous with the C-terminal part of the ERCC1 protein which, in a complex with XPF, is responsible for the 5'-incision reaction in human nucleotide excision repair. Both in the UvrC and the ERCC1 domain a Helix-hairpin-Helix (HhH) motif can be indicated, albeit at different positions. Such a motif also has been found in a large variety of DNA binding proteins and it has been suggested to form a structure involved in non-sequence-specific DNA binding. In contrast to the full length UvrC protein, a truncated UvrC protein (UvrC554) lacking the entire ERCC1 homology including the HhH motif no longer binds to ssDNA. Analysis of protein-DNA complexes using bandshift experiments showed that this putative DNA binding domain of UvrC is required for stabilisation of the UvrBC-DNA complex after the 3'-incision has taken place. We propose that after the initial 3'-incision the HhH motif recognises a specific DNA structure, thereby positioning the catalytic site for the subsequent 5'-incision reaction.     

The isomerization of the UvrB-DNA preincision complex couples the UvrB and UvrC activities

In Escherichia coli nucleotide excision repair, the UvrB-DNA preincision complex plays a key role, linking adduct recognition to incision. We previously showed that the efficiency of the incision is inversely related to the stability of the preincision complex. We postulated that an isomerization reaction converts [UvrB-DNA], stable but incompetent for incision, into the [UvrB-DNA]' complex, unstable and competent for incision. Here, we identify two parameters, negative supercoiling and presence of a nick at the fifth phosphodiester bond 3' to the lesion, that accelerate the isomerization leading to an increasing incision efficiency. We also show that the [UvrB-DNA] complex is more resistant to a salt concentration increase than the [UvrB-DNA]' complex. Finally, we report that the [UvrB-DNA]' is recognized by UvrC. These data suggest that the isomerization reaction leads to an exposure of single-stranded DNA around the lesion. This newly exposed single-stranded DNA serves as a binding site and substrate for the UvrC endonuclease. We propose that the isomerization reaction is responsible for coupling UvrB and UvrC activities and that this reaction corresponds to the binding of ATP.     

Protein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease.

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.     

The role of ATP binding and hydrolysis by UvrB during nucleotide excision repair

We have isolated UvrB-DNA complexes by capture of biotinylated damaged DNA substrates on streptavidin-coated magnetic beads. With this method the UvrB-DNA preincision complex remains stable even in the absence of ATP. For the binding of UvrC to the UvrB-DNA complex no cofactor is needed. The subsequent induction of 3' incision does require ATP binding by UvrB but not hydrolysis. This ATP binding induces a conformational change in the DNA, resulting in the appearance of the DNase I-hypersensitive site at the 5' side of the damage. In contrast, the 5' incision is not dependent on ATP binding because it occurs with the same efficiency with ADP. We show with competition experiments that both incision reactions are induced by the binding of the same UvrC molecule. A DNA substrate containing damage close to the 5' end of the damaged strand is specifically bound by UvrB in the absence of UvrA and ATP (Moolenaar, G. F., Monaco, V., van der Marel, G. A., van Boom, J. H., Visse, R., and Goosen, N. (2000) J. Biol. Chem. 275, 8038-8043). To initiate the formation of an active UvrBC-DNA incision complex, however, UvrB first needs to hydrolyze ATP, and subsequently a new ATP molecule must be bound. Implications of these findings for the mechanism of the UvrA-mediated formation of the UvrB-DNA preincision complex will be discussed.     

Formation and enzymatic properties of the UvrB.DNA complex

The UvrA, UvrB, and UvrC proteins collectively catalyze the dual incision of a damaged DNA strand in an ATP-dependent reaction. We previously reported (Orren, D. K., and Sancar, A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5237-5241) that UvrA delivers UvrB to damaged sites in DNA; upon addition of UvrC to these UvrB.DNA complexes, the DNA is incised. In the present study, we have further characterized both the delivery of UvrB to DNA and the subsequent incision process, with emphasis on the role of ATP in these reactions. The UvrA-dependent delivery of UvrB onto damaged DNA is relatively slow (kon approximately 6 x 10(4) M-1 s-1) and requires ATP hydrolysis (Km = 120 microM). Although ATP enhances the stability of UvrB.DNA complexes (koff = 8.5 x 10(-5) s-1), the isolated UvrB.DNA complexes do not contain any covalently attached or stably bound nucleotide. However, ATP binding is required for the UvrC-dependent dual incision of DNA bound by UvrB. Interestingly, adenosine 5'-(3-O-thio)triphosphate can substitute for ATP at this step. The Km for ATP during incision is 2 microM, but ATP is not hydrolyzed at a detectable level during the incision reaction. The incisions made by UvrB-UvrC are on both sides of the adduct and result in the excision of the damaged nucleotide.     

Role of the Escherichia coli nucleotide excision repair proteins in DNA replication

DNA polymerase I (PolI) functions both in nucleotide excision repair (NER) and in the processing of Okazaki fragments that are generated on the lagging strand during DNA replication. Escherichia coli cells completely lacking the PolI enzyme are viable as long as they are grown on minimal medium. Here we show that viability is fully dependent on the presence of functional UvrA, UvrB, and UvrD (helicase II) proteins but does not require UvrC. In contrast, delta polA cells grow even better when the uvrC gene has been deleted. Apparently UvrA, UvrB, and UvrD are needed in a replication backup system that replaces the PolI function, and UvrC interferes with this alternative replication pathway. With specific mutants of UvrC we could show that the inhibitory effect of this protein is related to its catalytic activity that on damaged DNA is responsible for the 3' incision reaction. Specific mutants of UvrA and UvrB were also studied for their capacity to support the PolI-independent replication. Deletion of the UvrC-binding domain of UvrB resulted in a phenotype similar to that caused by deletion of the uvrC gene, showing that the inhibitory incision activity of UvrC is mediated via binding to UvrB. A mutation in the N-terminal zinc finger domain of UvrA does not affect NER in vivo or in vitro. The same mutation, however, does give inviability in combination with the delta polA mutation. Apparently the N-terminal zinc-binding domain of UvrA has specifically evolved for a function outside DNA repair. A model for the function of the UvrA, UvrB, and UvrD proteins in the alternative replication pathway is discussed.     

Oligomerization of the UvrB nucleotide excision repair protein of Escherichia coli.

A combination of hydrodynamic and cross-linking studies were used to investigate self-assembly of the Escherichia coli DNA repair protein UvrB. Though the procession of steps leading to incision of DNA at sites flanking damage requires that UvrB engage in an ordered series of complexes, successively with UvrA, DNA, and UvrC, the potential for self-association had not yet been reported. Gel permeation chromatography, nondenaturing polyacrylamide gel electrophoresis, and chemical cross-linking results combine to show that UvrB stably assembles as a dimer in solution at concentrations in the low micromolar range. Smaller populations of higher order oligomeric species are also observed. Unlike the dimerization of UvrA, an initial step promoted by ATP binding, the monomer-dimer equilibrium for UvrB is unaffected by the presence of ATP. The insensitivity of cross-linking efficiency to a 10-fold variation in salt concentration further suggests that UvrB self-assembly is driven largely by hydrophobic interactions. Self-assembly is significantly weakened by proteolytic removal of the carboxyl terminus of the protein (generating UvrB*), a domain also known to be required for the interaction with UvrC leading to the initial incision of damaged DNA. This suggests that the C terminus may be a multifunctional binding domain, with specificity regulated by protein conformation.     

Active site of (A)BC excinuclease. I. Evidence for 5' incision by UvrC through a catalytic site involving Asp399, Asp438, Asp466, and His538 residues.

(A)BC excinuclease of Escherichia coli removes damaged nucleotides from DNA by hydrolyzing the 8th phosphodiester bond 5' and the 15th phosphodiester bond 3' to the modified base. The activity results from the ordered action of UvrA, UvrB, and UvrC proteins. The role of UvrA is to help assemble the UvrB.DNA complex, and it is not involved in the actual incision reactions which are carried out by UvrB and UvrC. To investigate the role of UvrC in the nuclease activity a subset of His, Asp, and Glu residues in the C-terminal half of the protein were mutagenized in vitro. The effect of these mutations on UV resistance in vivo and incision activity in vitro were investigated. Mutations, H538F, D399A, D438A, and D466A conferred extreme UV sensitivity. Enzyme reconstituted with these mutant proteins carried out normal 3' incision but was completely defective in 5' incision activity. Our data suggest that UvrC makes the 5' incision by employing a mechanism whereby the three carboxylates acting in concert with H538 and a Mg2+ ion facilitate nucleophilic attack by an active site water molecule.