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.     

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.     

The beta -hairpin motif of UvrB is essential for DNA binding, damage processing, and UvrC-mediated incisions.

UvrB plays a major role in recognition and processing of DNA lesions during nucleotide excision repair. The crystal structure of UvrB revealed a similar fold as found in monomeric DNA helicases. Homology modeling suggested that the beta-hairpin motif of UvrB might be involved in DNA binding (Theis, K., Chen, P. J., Skorvaga, M., Van Houten, B., and Kisker, C. (1999) EMBO J. 18, 6899-6907). To determine a role of the beta-hairpin of Bacillus caldotenax UvrB, we have constructed a deletion mutant, Deltabetah UvrB, which lacks residues Gln-97-Asp-112 of the beta-hairpin. Deltabetah UvrB does not form a stable UvrB-DNA pre-incision complex and is inactive in UvrABC-mediated incision. However, Deltabetah UvrB is able to bind to UvrA and form a complex with UvrA and damaged DNA, competing with wild type UvrB. In addition, Deltabetah UvrB shows wild type-like ATPase activity in complex with UvrA that is stimulated by damaged DNA. In contrast to wild type UvrB, the ATPase activity of mutant UvrB does not lead to a destabilization of the damaged duplex. These results indicate that the conserved beta-hairpin motif is a major factor in DNA binding.     

The presence of two UvrB subunits in the UvrAB complex ensures damage detection in both DNA strands

It is generally accepted that the damage recognition complex of nucleotide excision repair in Escherichia coli consists of two UvrA and one UvrB molecule, and that in the preincision complex UvrB binds to the damage as a monomer. Using scanning force microscopy, we show here that the damage recognition complex consists of two UvrA and two UvrB subunits, with the DNA wrapped around one of the UvrB monomers. Upon binding the damage and release of the UvrA subunits, UvrB remains a dimer in the preincision complex. After association with the UvrC protein, one of the UvrB monomers is released. We propose a model in which the presence of two UvrB subunits ensures damage recognition in both DNA strands. Upon binding of the UvrA(2)B(2) complex to a putative damaged site, the DNA wraps around one of the UvrB monomers, which will subsequently probe one of the DNA strands for the presence of a lesion. When no damage is found, the DNA will wrap around the second UvrB subunit, which will check the other strand for aberrations.     

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.     

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.     

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.     

Binding of the UvrB dimer to non-damaged and damaged DNA: residues Y92 and Y93 influence the stability of both subunits

UvrB is the ultimate damage-binding protein in bacterial nucleotide excision repair. Previous AFM experiments have indicated that UvrB binds to a damage as a dimer. In this paper we visualize for the first time a UvrB dimer in a gel retardation assay, with the second subunit (B2) more loosely bound than the subunit (B1) that interacts with the damage. A beta-hairpin motif in UvrB plays an important role in damage specific binding. Alanine substitutions of Y92 or Y93 in the beta-hairpin result in proteins that kill E. coli cells as a consequence of incision in non-damaged DNA. Apparently, both residues are needed to prevent binding of UvrB to non-damaged DNA. The lethality of Y93A results from UvrC-mediated incisions, whereas that of Y92A is due to incisions by Cho. This difference could be ascribed to a difference in stability of the B2 subunit in the mutant UvrB-DNA complexes. We show that for 3' incision UvrC needs to displace this second UvrB subunit from the complex, whereas Cho seems capable to incise the dimer-complex. Footprint analysis of the contacts of UvrB with damaged DNA revealed that the B2 subunit interacts with the flanking DNA at the 3' side of the lesion. The B2 subunit of mutant Y92A appeared to be more firmly associated with the DNA, indicating that even when B1 is bound to a lesion, the B2 subunit probes the adjacent DNA for presence of damage. We propose this to be a reflection of the process that the UvrB dimer uses to find lesions in the DNA. In addition to preventing binding to non-damaged DNA, the Y92 and Y93 residues appear also important for making specific contacts (of B1) with the damaged site. We show that the concerted action of the two tyrosines lead to a conformational change in the DNA surrounding the lesion, which is required for the 3' incision reaction.     

UvrB domain 4, an autoinhibitory gate for regulation of DNA binding and ATPase activity

UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA. Regulation of DNA binding and ATP hydrolysis by UvrB is poorly understood. Using atomic force microscopy and biochemical assays, we found that truncation of domain 4 of Bacillus caldotenax UvrB (UvrBDelta4) leads to multiple changes in protein function. Protein dimerization decreases with an approximately 8-fold increase of the equilibrium dissociation constant and an increase in DNA binding. Loss of domain 4 causes the DNA binding mode of UvrB to change from dimer to monomer, and affinity increases with the apparent dissociation constants on nondamaged and damaged single-stranded DNA decreasing 22- and 14-fold, respectively. ATPase activity by UvrBDelta4 increases 14- and 9-fold with and without single-stranded DNA, respectively, and UvrBDelta4 supports UvrA-independent damage-specific incision by Cho on a bubble DNA substrate. We propose that other than its previously discovered role in regulating protein-protein interactions, domain 4 is an autoinhibitory domain regulating the DNA binding and ATPase activities of UvrB.     

Functions of base flipping in E. coli nucleotide excision repair

UvrB is the main damage recognition protein in bacterial nucleotide excision repair and is capable of recognizing various structurally unrelated types of damage. Previously we have shown that upon binding of Escherichia coli UvrB to damaged DNA two nucleotides become extrahelical: the nucleotide directly 3' to the lesion and its base-pairing partner in the non-damaged strand. Here we demonstrate using a novel fluorescent 2-aminopurine-menthol modification that the position of the damaged nucleotide itself does not change upon UvrB binding. A co-crystal structure of B. caldotenax UvrB and DNA has revealed that one nucleotide is flipped out of the DNA helix into a pocket of the UvrB protein where it stacks on Phe249 [J.J. Truglio, E. Karakas, B. Hau, H. Wang, M.J. DellaVecchia, B. van Houten, C. Kisker, Structural basis for DNA recognition and processing by UvrB, Nat. Struct. Mol. Biol. 13 (2006) 360-364]. By mutating the equivalent of Phe249 (Tyr249) in the E. coli UvrB protein we show that on damaged DNA neither of the extrahelical nucleotides is inserted into this protein pocket. The mutant UvrB protein, however, resulted in an increased binding and incision of undamaged DNA showing that insertion of a base into the nucleotide-binding pocket is important for dissociation of UvrB from undamaged sites. Replacing the nucleotides in the non-damaged strand with a C3-linker revealed that the extruded base in the non-damaged strand is not directly involved in UvrB-binding or UvrC-mediated incision, but that its displacement is needed to allow access for residues of UvrB or UvrC to the neighboring base, which is directly opposite the DNA damage. This interaction is shown to be essential for optimal 3'-incision by UvrC. After 3'-incision base flipping in the non-damaged DNA strand is lost, indicative for a conformational change needed to prepare the UvrB-DNA complex for 5'-incision.     

Crystal structure of UvrB, a DNA helicase adapted for nucleotide excision repair

Nucleotide excision repair (NER) is a highly conserved DNA repair mechanism. NER systems recognize the damaged DNA strand, cleave it on both sides of the lesion, remove and newly synthesize the fragment. UvrB is a central component of the bacterial NER system participating in damage recognition, strand excision and repair synthesis. We have solved the crystal structure of UvrB in the apo and the ATP-bound forms. UvrB contains two domains related in structure to helicases, and two additional domains unique to repair proteins. The structure contains all elements of an intact helicase, and is evidence that UvrB utilizes ATP hydrolysis to move along the DNA to probe for damage. The location of conserved residues and structural comparisons allow us to predict the path of the DNA and suggest that the tight pre-incision complex of UvrB and the damaged DNA is formed by insertion of a flexible beta-hairpin between the two DNA strands.     

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.     

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.     

Analyzing the handoff of DNA from UvrA to UvrB utilizing DNA-protein photoaffinity labeling

To better define the molecular architecture of nucleotide excision repair intermediates it is necessary to identify the specific domains of UvrA, UvrB, and UvrC that are in close proximity to DNA damage during the repair process. One key step of nucleotide excision repair that is poorly understood is the transfer of damaged DNA from UvrA to UvrB, prior to incision by UvrC. To study this transfer, we have utilized two types of arylazido-modified photoaffinity reagents that probe residues in the Uvr proteins that are closest to either the damaged or non-damaged strands. The damaged strand probes consisted of dNTP analogs linked to a terminal arylazido moiety. These analogs were incorporated into double-stranded DNA using DNA polymerase beta and functioned as both the damage site and the cross-linking reagent. The non-damaged strand probe contained an arylazido moiety coupled to a phosphorothioate-modified backbone of an oligonucleotide opposite the damaged strand, which contained an internal fluorescein adduct. Six site-directed mutants of Bacillus caldotenax UvrB located in different domains within the protein (Y96A, E99A, R123A, R183E, F249A, and D510A), and two domain deletions (Delta2 and Deltabeta-hairpin), were assayed. Data gleaned from these mutants suggest that the handoff of damaged DNA from UvrA to UvrB proceeds in a three-step process: 1) UvrA and UvrB bind to the damaged site, with UvrA in direct contact; 2) a transfer reaction with UvrB contacting mostly the non-damaged DNA strand; 3) lesion engagement by the damage recognition pocket of UvrB with concomitant release of UvrA.     

Clue to damage recognition by UvrB: residues in the beta-hairpin structure prevent binding to non-damaged DNA.

UvrB, the ultimate damage-recognizing component of bacterial nucleotide excision repair, contains a flexible beta-hairpin rich in hydrophobic residues. We describe the properties of UvrB mutants in which these residues have been mutated. The results show that Y101 and F108 in the tip of the hairpin are important for the strand-separating activity of UvrB, supporting the model that the beta-hairpin inserts between the two DNA strands during the search for DNA damage. Residues Y95 and Y96 at the base of the hairpin have a direct role in damage recognition and are positioned close to the damage in the UvrB-DNA complex. Strikingly, substituting Y92 and Y93 results in a protein that is lethal to the cell. The mutant protein forms pre- incision complexes on non-damaged DNA, indicating that Y92 and Y93 function in damage recognition by preventing UvrB binding to non-damaged sites. We propose a model for damage recognition by UvrB in which, stabilized by the four tyrosines at the base of the hairpin, the damaged nucleotide is flipped out of the DNA helix.     

Thermodynamic characterization of the interaction of mutant UvrB protein with damaged DNA

During the DNA damage recognition of nucleotide excision repair in Escherichia coli the interaction of UvrB protein with damaged DNA ensures the recognition of differences in the intrinsic chemical structures of a variety of adduct molecules in DNA double helix. Our earlier study indicated that a single tyrosine-to-tryptophan mutation at residue 95 converted the UvrB to a protein [UvrB(Y95W)] that is able to bind to a structure-specific bubble DNA substrate, even in the absence of UvrA. Fluorescence spectroscopy therefore was adopted to investigate the biochemical properties and thermodynamics of DNA damage recognition by the mutant protein. We examined the binding of the UvrB(Y95W) mutant protein to a structure-specific 30 bp DNA substrate containing a single fluorescein which serves as both an adduct and a fluorophore. Binding of the protein to the substrate results in a significant reduction in fluorescence. By monitoring the fluorescence changes, binding isotherms were generated from a series of titration experiments at various physiological temperatures, and dissociation constants were determined. Analysis of our data indicate that interaction of UvrB(Y95W) protein with the adduct incurred a large negative change in heat capacity DeltaC(p)(o)(obs) (-1.1 kcal mol(-1) K(-1)), while the DeltaG(o)(obs) was relatively unchanged with temperature. Further study of the binding at various concentrations of KCl showed that on average only about 1.5 ion pairs were involved in formation of the UvrB-DNA complex. Together, these results suggested that hydrophobic interactions are the main driving forces for the recognition of DNA damage by UvrB protein.     

DNA damage recognition of mutated forms of UvrB proteins in nucleotide excision repair

The DNA repair protein UvrB plays an indispensable role in the stepwise and sequential damage recognition of nucleotide excision repair in Escherichia coli. Our previous studies suggested that UvrB is responsible for the chemical damage recognition only upon a strand opening mediated by UvrA. Difficulties were encountered in studying the direct interaction of UvrB with adducts due to the presence of UvrA. We report herein that a single point mutation of Y95W in which a tyrosine is replaced by a tryptophan results in an UvrB mutant that is capable of efficiently binding to structure-specific DNA adducts even in the absence of UvrA. This mutant is fully functional in the UvrABC incisions. The dissociation constant for the mutant-DNA adduct interaction was less than 100 nM at physiological temperatures as determined by fluorescence spectroscopy. In contrast, similar substitutions at other residues in the beta-hairpin with tryptophan or phenylalanine do not confer UvrB such binding ability. Homology modeling of the structure of E. coli UvrB shows that the aromatic ring of residue Y95 and only Y95 directly points into the DNA binding cleft. We have also examined UvrB recognition of both "normal" bulky BPDE-DNA and protein-cross-linked DNA (DPC) adducts and the roles of aromatic residues of the beta-hairpin in the recognition of these lesions. A mutation of Y92W resulted in an obvious decrease in the efficiency of UvrABC incisions of normal adducts, while the incision of the DPC adduct is dramatically increased. Our results suggest that Y92 may function differently with these two types of adducts, while the Y95 residue plays an unique role in stabilizing the interaction of UvrB with DNA damage, most likely by a hydrophobic stacking.     

Identification of residues within UvrB that are important for efficient DNA binding and damage processing

The UvrB protein is the central recognition protein in bacterial nucleotide excision repair. We have shown previously that the highly conserved beta-hairpin motif in Bacillus caldotenax UvrB is essential for DNA binding, damage recognition, and UvrC-mediated incision, as deletion of the upper part of the beta-hairpin (residues 97-112) results in the inability of UvrB to be loaded onto damaged DNA, defective incision, and the lack of strand-destabilizing activity. In this work, we have further examined the role of the beta-hairpin motif of UvrB by a mutational analysis of 13 amino acids within or in the vicinity of the beta-hairpin. These amino acids are predicted to be important for the interaction of UvrB with both damaged and non-damaged DNA strands as well as the formation of salt bridges between the beta-hairpin and domain 1b of UvrB. The resulting mutants were characterized by standard functional assays such as oligonucleotide incision, electrophoretic mobility shift, strand-destabilizing, and ATPase assays. Our data indicated a direct role of Tyr96, Glu99, and Arg123 in damage-specific DNA binding. In addition, Tyr93 plays an important but less essential role in DNA binding by UvrB. Finally, the formation of salt bridges between the beta-hairpin and domain 1b, involving amino acids Lys111 bound to Glu307 and Glu99 bound to Arg367 or Arg289, are important but not essential for the function of UvrB.     

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.     

Structure and function of the (A)BC excinuclease of Escherichia coli

(A)BC excinuclease is the enzymatic activity resulting from the mixture of E. coli UvrA, UvrB and UvrC proteins with damaged DNA. This is a functional definition as new evidence suggests that the three proteins never associate in a ternary complex. The UvrA subunit associates with the UvrB subunit in the form of an A2B1 complex which, guided by UvrA's affinity for damaged DNA binds to a lesion in DNA and delivers the UvrB subunit to the damaged site. The UvrB-damaged DNA complex is extremely stable (t1/2 congruent to 100 min). The UvrC subunit, which has no specific affinity for damaged DNA, recognizes the UvrB-DNA complex with high specificity and the protein complex consisting of UvrB and UvrC proteins makes two incisions, the 8th phosphodiester bond 5' and the 5th phosphodiester bond 3' to the damaged nucleotide. (A)BC excinuclease recognizes DNA damage ranging from AP sites and thymine glycols to pyrimidine dimers, and the adducts of psoralen, cisplatinum, mitomycin C, 4-nitroquinoline oxide and interstrand crosslinks.