Deletion mutagenesis of the Escherichia coli UvrA protein localizes domains for DNA binding, damage recognition, and protein-protein interactions.

The UvrA protein is the DNA binding and damage recognition subunit of the damage-specific UvrABC endonuclease. In addition, it is an ATPase/GTPase, and the binding energy of ATP is linked to dimerization of the UvrA protein. Furthermore, the UvrA protein interacts with the UvrB protein to modulate its activities, both in solution and in association with DNA, where the UvrAB complex possesses a helicase activity. The domains of the UvrA protein that sponsor each of these activities were localized within the protein by studying the in vitro properties of a set of purified deletion mutants of the UvrA protein. A region located within the first 230 amino acids was found to contain the minimal region necessary for interactions with UvrB, the UvrA dimerization interface was localized to within the first 680 amino acids, and the DNA binding domain lies within the first 900 amino acids of the 940-amino acid UvrA protein. Two damage recognition domains were detected. The first domain, which coincides with the DNA binding region, is required to detect the damage. The second domain, located on or near the C-terminal 40 amino acids, stabilizes the protein-DNA complex when damage is encountered.     

Characterization of the helicase activity of the Escherichia coli UvrAB protein complex

The requirement for nucleotide hydrolysis in the DNA repair mechanism of the Escherichia coli UvrABC protein complex has been analyzed. The DNA-activated UvrAB ATPase activity is part of a helicase activity exhibited by the UvrAB protein complex. The helicase acts only on short duplexes and, therefore, is unlike other helicases such as those involved in DNA replication that unwind long duplexes. The strand displacement activity occurs in the 5'----3' direction and requires either ATP or dATP. The helicase activity is inhibited by UV photoproducts. The absence of this activity in a complex formed with proteolyzed UvrB (UvrB*), a complex also deficient in the endonuclease activity, suggests that this activity is important in the repair mechanism. The UvrAB protein complex may remain bound to a damaged site and by coupling the energy derived from ATP hydrolysis, alter the DNA conformation around the damage site to one that is permissive for endonucleolytic events. The conformational changes may take the form of DNA unwinding.     

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.     

Site-specific mutagenesis of conserved residues within Walker A and B sequences of Escherichia coli UvrA protein.

UvrA is the ATPase subunit of the DNA repair enzyme (A)BC excinuclease. The amino acid sequence of this protein has revealed, in addition to two zinc fingers, three pairs of nucleotide binding motifs each consisting of a Walker A and B sequence. We have conducted site-specific mutagenesis, ATPase kinetic analyses, and nucleotide binding equilibrium measurements to correlate these sequence motifs with activity. Replacement of the invariant Lys by Ala in the putative A sequences indicated that K37 and K646 but not K353 are involved in ATP hydrolysis. In contrast, substitution of the invariant Asp by Asn in the B sequences at positions D238, D513, or D857 had little effect on the in vivo activity of the protein. Nucleotide binding studies revealed a stoichiometry of 0.5 ADP/UvrA monomer while kinetic measurements on wild-type and mutant proteins showed that the active form of UvrA is a dimer with 2 catalytic sites which interact in a positive cooperative manner in the presence of ADP; mutagenesis of K37 but not of K646 attenuated this cooperativity. Loss of ATPase activity was about 75% in the K37A, 86% in the K646A mutant, and 95% in the K37A-K646A double mutant. These amino acid substitutions had only a marginal effect on the specific binding of UvrA to damaged DNA but drastically reduced its ability to deliver UvrB to the damage site. We find that the deficient UvrB loading activity of these mutant UvrA proteins results from their inability to associate with UvrB in the form of (UvrA)2(UvrB)1 complexes. We conclude that UvrA forms a dimer with two ATPase domains involving K37 and K646 and that the work performed by ATP hydrolysis is the delivery of UvrB to the damage site on DNA.     

Role of the insertion domain and the zinc-finger motif of Escherichia coli UvrA in damage recognition and ATP hydrolysis

UvrA is the initial DNA damage-sensing protein in bacterial nucleotide excision repair. Each protomer of the UvrA dimer contains two ATPase domains, that belong to the family of ATP-binding cassette domains. Three structural domains are inserted in these ATPase domains: the insertion domain (ID) and UvrB binding domain (in ATP domain I) and the zinc-finger motif (in ATP domain II). In this paper we analyze the function of the ID and the zinc finger motif in damage specific binding of Escherichia coli UvrA. We show that the ID is not essential for damage discrimination, but it does stabilize UvrA on the DNA, most likely by forming a clamp around the DNA helix. We present evidence that two conserved arginine residues in the ID contact the phosphate backbone of the DNA, leading to strand separation after the ATPase-driven movement of the ID's. Remarkably, deletion of the ID generated a phenotype in which UV-survival strongly depends on the presence of photolyase, indicating that UvrA and photolyase form a ternary complex on a CPD-lesion. The zinc-finger motif is shown to be important for the transfer of the damage recognition signal to the ATPase of UvrA. In the absence of this domain the coupling between DNA binding and ATP hydrolysis is completely lost. Mutation of the phenylalanine residue in the tip of the zinc-finger domain resulted in a protein in which the ATPase was already triggered when binding to an undamaged site. As the zinc-finger motif is connected to the DNA binding regions on the surface of UvrA, this strongly suggests that damage-specific binding to these regions results in a rearrangement of the zinc-finger motif, which in its turn activates the ATPase. We present a model how damage recognition is transmitted to activate ATP hydrolysis in ATP binding domain I of the protein.     

Steady-state kinetic analysis of ATP hydrolysis by the B protein of bacteriophage mu. Involvement of protein oligomerization in the ATPase cycle

The DNA strand-transfer reaction of bacteriophage Mu requires Mu B protein and ATP for high efficiency. These factors facilitate the capture of target DNA by the donor protein-DNA complex. To understand the mechanism of the Mu B ATPase cycle in the Mu DNA strand-transfer reaction, we undertook a steady-state kinetic analysis of Mu B ATPase. The results reveal complex properties of the ATPase activity; Mu B protein oligomerizes in the presence of ATP, and ATP hydrolysis by the Mu B ATPase is stimulated by the protein oligomerization and shows a positive cooperativity with respect to ATP concentration. Mu B ATPase activity is also modulated by DNA and Mu A protein. DNA alone suppresses the catalytic activity of Mu B ATPase, whereas DNA enhances the apparent binding affinity for ATP. In the presence of Mu A protein together with DNA, however, the catalytic activity is greatly stimulated. Based on these results, we propose a working hypothesis in which oligomerization of Mu B protein plays a key role in its ATPase cycle.     

Isolation and characterization of functional domains of UvrA.

The sequence of Escherichia coli UvrA protein suggests that it may fold into two functional domains each possessing DNA binding and ATPase activities. We have taken two approaches to physically isolate polypeptides corresponding to the two putative domains. First, a 180 base pair DNA segment encoding multiple collagenase recognition sequences was inserted into UvrA's putative interdomain hinge region. This UvrA derivative was purified and digested with collagenase, and the resulting 70-kDa N-terminal and 35-kDa C-terminal fragments were purified. Both fragments possessed nonspecific DNA binding activity, but only the N-terminal domain retained its nucleotide binding capacity as evidence by measurements of ATP hydrolysis and by ATP photo-cross-linking. Together, the two fragments failed to substitute for UvrA in reconstituting (A)BC excinuclease and, therefore, were presumed to be unable to load UvrB onto damaged DNA. Second, the DNA segments encoding the two domains were fused to the beta-galactosidase gene. The UvrA N-terminal domain-beta-galactosidase fusion protein was overproduced and purified. This fusion protein had ATPase activity, thus confirming that the amino-terminal domain does possess an intrinsic ATPase activity independent of any interaction with the carboxy terminus. Our results show that UvrA has two functional domains and that the specificity for binding to damaged DNA is provided by the proper three-dimensional orientation of one zinc finger motif relative to the other and is not an intrinsic property of an individual zinc finger domain.     

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.     

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 aparently 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.     

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.     

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.     

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.     

Interaction of UvrA and UvrB proteins with a fluorescent single-stranded DNA. Implication for slow conformational change upon interaction of UvrB with DNA

UvrA and UvrB proteins play key roles in the damage recognition step in the nucleotide excision repair. However, the molecular mechanism of damage recognition by these proteins is still not well understood. In this work we analyzed the interaction between single-stranded DNA (ssDNA) labeled with a fluorophore tetramethylrhodamine (TMR) and Thermus thermophilus HB8 UvrA (ttUvrA) and UvrB (ttUvrB) proteins. TMR-labeled ssDNA (TMR-ssDNA) as well as UV-irradiated ssDNA stimulated ATPase activity of ttUvrB more strongly than did normal ssDNA, indicating that this fluorescent ssDNA was recognized as damaged ssDNA. The addition of ttUvrA or ttUvrB enhanced the fluorescence intensity of TMR-ssDNA, and the intensity was much greater in the presence of ATP. Fluorescence titration indicated that ttUvrA has higher specificity for TMR-ssDNA than for normal ssDNA in the absence of ATP. The ttUvrB showed no specificity for TMR-ssDNA, but it took over 200 min for the fluorescence intensity of the ttUvrB-TMR-ssDNA complex to reach saturation in the presence of ATP. This time-dependent change could be separated into two phases. The first phase was rapid, whereas the second phase was slow and dependent on ATP hydrolysis. Time dependence of ATPase activity and fluorescence polarization suggested that changes other than the binding reaction occurred during the second phase. These results strongly suggest that ttUvrB binds ssDNA quickly and that a conformational change in ttUrvB-ssDNA complex occurs slowly. We also found that DNA containing a fluorophore as a lesion is useful for directly investigating the damage recognition by UvrA and UvrB.     

Both ATPase sites of Escherichia coli UvrA have functional roles in nucleotide excision repair.

The roles of the two tandemly arranged putative ATP binding sites of Escherichia coli UvrA in UvrABC endonuclease-mediated excision repair were analyzed by site-directed mutagenesis and biochemical characterization of the representative mutant proteins. Evidence is presented that UvrA has two functional ATPase sites which coincide with the putative ATP binding motifs predicted from its amino acid sequence. The individual ATPase sites can independently hydrolyze ATP. The C-terminal ATPase site has a higher affinity for ATP than the N-terminal site. The invariable lysine residues at the ends of the glycine-rich loops of the consensus Walker type "A" motifs are indispensable for ATP hydrolysis. However, the mutations at these lysine residues do not significantly affect ATP binding. UvrA, with bound ATP, forms the most favored conformation for DNA binding. The initial binding of UvrA to DNA is chiefly at the undamaged sites. In contrast to the wild type UvrA, the ATPase site mutants bind equally to damaged and undamaged sites. Dissociation of tightly bound nucleoprotein complexes from the undamaged sites requires hydrolysis of ATP by the C-terminal ATPase site of UvrA. Thus, both ATP binding and hydrolysis are required for the damage recognition step enabling UvrA to discriminate between damaged and undamaged sites on DNA.     

The UvrABC endonuclease system of Escherichia coli--a view from Baltimore

Nucleotide excision is initiated by the UvrABC endonuclease system in which the initial DNA interaction is with UvrA which was dimerized in the presence of ATP. Nucleoprotein formation most likely takes place on undamaged regions of DNA by (UvrA)2 which has been dimerized in the presence of ATP. Topological unwinding of DNA, driven by ATP binding, is increased by the presence of UvrB to approximately a single helical turn. The Uvr(A)2B complex translocates to a damaged site by the combined Uvr(A)2B helicase in which the driving force is provided by the UvrB-associated ATPase. The dual incision reaction is initiated by the binding of the UvrC protein to the Uvr(A)2B-nucleoprotein complex. The proteins in this post-incision nucleoprotein complex do not turn over and require the presence of the UvrD protein and DNA polymerase I under polymerizing conditions. The final integrity of the DNA strands is restored with polynucleotide ligase.     

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.     

Role of the two ATPase domains of Escherichia coli UvrA in binding non-bulky DNA lesions and interaction with UvrB

The UvrA protein is the initial DNA damage-sensing protein in bacterial nucleotide excision repair and detects a wide variety of structurally unrelated lesions. After initial recognition of DNA damage, UvrA loads the UvrB protein onto the DNA. This protein then verifies the presence of a lesion, after which UvrA is released from the DNA.UvrA contains two ATPase domains, both belonging to the ABC ATPase superfamily. We have determined the activities of two mutants, in which a single domain was deactivated. Inactivation of either one ATPase domain in Escherichia coli UvrA results in a complete loss of ATPase activity, indicating that both domains function in a cooperative way. We could show that this ATPase activity is not required for the recognition of bulky lesions by UvrA, but it does promote the specific binding to the less distorting cyclobutane–pyrimidine dimer (CPD). The two ATPase mutants also show a difference in UvrB-loading, depending on the length of the DNA substrate. The ATPase domain I mutant was capable of loading UvrB on a lesion in a 50 bp fragment, but this loading was reduced on a longer substrate. For the ATPase domain II mutant the opposite was found: UvrB could not be loaded on a 50 bp substrate, but this loading was rescued when the length of the fragment was increased. This differential loading of UvrB by the two ATPase mutants could be related to different interactions between the UvrA and UvrB subunits.     

Analysis of sequential steps of nucleotide excision repair in Escherichia coli using synthetic substrates containing single psoralen adducts

Escherichia coli ABC excinuclease initiates the removal of dodecanucleotides from damaged DNA in an ATP-dependent reaction. Using a synthetic DNA fragment containing a psoralen adduct at a defined position we have investigated the interaction of the components of the enzyme with substrate by DNase I footprinting. We find that the UvrA subunit binds to DNA specifically in the absence of cofactors and that the binding affinity is stimulated about 4-fold by ATP and only marginally inhibited by ADP. The UvrA.DNA complexes formed in the absence of co-factors or in the presence of either ATP or ADP are remarkably similar. In contrast, adenosine 5'-O-(thiotriphosphate) increases nonspecific binding and completely abolishes the UvrA footprint. The UvrB subunit can associate with the UvrA subunit on DNA in the absence of ATP, but this ternary UvrA.UvrB.DNA complex is qualitatively different from that formed in the presence of ATP. The UvrC subunit elicits no additional change in the UvrA-UvrB footprint. Helicase II (UvrD protein) does not alter the UvrA-UvrB footprint but does appear to interact at the 5'-incision site of the postincision complex. DNA polymerase I fills in the excision gap in the presence or absence of helicase II and apparently releases the ABC excinuclease from the repaired DNA. Nearly 90% of the repair patches are 12 nucleotides long, and this length is not affected by helicase II. We see no evidence by DNase I footprinting for the formation of a multiprotein complex encompassing the UvrA, -B, -C, and -D proteins and DNA polymerase I.     

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.