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.     

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.     

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.     

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.     

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.     

Architecture of nucleotide excision repair complexes: DNA is wrapped by UvrB before and after damage recognition

Nucleotide excision repair (NER) is a major DNA repair mechanism that recognizes a broad range of DNA damages. In Escherichia coli, damage recognition in NER is accomplished by the UvrA and UvrB proteins. We have analysed the structural properties of the different protein-DNA complexes formed by UvrA, UvrB and (damaged) DNA using atomic force microscopy. Analysis of the UvrA(2)B complex in search of damage revealed the DNA to be wrapped around the UvrB protein, comprising a region of about seven helical turns. In the UvrB-DNA pre-incision complex the DNA is wrapped in a similar way and this DNA configuration is dependent on ATP binding. Based on these results, a role for DNA wrapping in damage recognition is proposed. Evidence is presented that DNA wrapping in the pre-incision complex also stimulates the rate of incision by UvrC.     

Crystal structure of Thermus thermophilus HB8 UvrB protein, a key enzyme of nucleotide excision repair

In the nucleotide excision repair system, UvrB plays a central role in damage recognition and DNA incision by interacting with UvrA and UvrC. We have determined the crystal structure of Thermus thermophilus HB8 UvrB at 1.9 A resolution. UvrB comprises four domains, two of which have an alpha/beta structure resembling the core domains of DNA and RNA helicases. Additionally, UvrB has an alpha-helical domain and a domain consisting of antiparallel beta-sheets (beta-domain). The sequence similarity suggests that the beta-domain interacts with UvrA. Based on the distribution of the conserved regions and the structure of the PcrA-DNA complex, a model for the UvrB-DNA complex is proposed.     

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.     

Interactions between UvrA and UvrB: the role of UvrB's domain 2 in nucleotide excision repair

Nucleotide excision repair (NER) is a highly conserved DNA repair mechanism present in all kingdoms of life. UvrB is a central component of the bacterial NER system, participating in damage recognition, strand excision and repair synthesis. None of the three presently available crystal structures of UvrB has defined the structure of domain 2, which is critical for the interaction with UvrA. We have solved the crystal structure of the UvrB Y96A variant, which reveals a new fold for domain 2 and identifies highly conserved residues located on its surface. These residues are restricted to the face of UvrB important for DNA binding and may be critical for the interaction of UvrB with UvrA. We have mutated these residues to study their role in the incision reaction, formation of the pre-incision complex, destabilization of short duplex regions in DNA, binding to UvrA and ATP hydrolysis. Based on the structural and biochemical data, we conclude that domain 2 is required for a productive UvrA-UvrB interaction, which is a pre-requisite for all subsequent steps in nucleotide excision repair.     

The C-terminal zinc finger of UvrA does not bind DNA directly but regulates damage-specific DNA binding

In prokaryotic nucleotide excision repair, UvrA recognizes DNA perturbations and recruits UvrB for the recognition and processing steps in the reaction. One of the most remarkable aspects of UvrA is that it can recognize a wide range of DNA lesions that differ in chemistry and structure. However, how UvrA interacts with DNA is unknown. To examine the role that the UvrA C-terminal zinc finger domain plays in DNA binding, an eleven amino acid deletion was constructed (ZnG UvrA). Biochemical characterization of the ZnG UvrA protein was carried out using UvrABC DNA incision, DNA binding and ATPase assays. Although ZnG UvrA was able to bind dsDNA slightly better than wild-type UvrA, the ZnG UvrA mutant only supported 50-75% of wild type incision. Surprisingly, the ZnG UvrA mutant, while retaining its ability to bind dsDNA, did not support damage-specific binding. Furthermore, this mutant protein only provided 10% of wild-type Bca UvrA complementation for UV survival of an uvrA deletion strain. In addition, ZnG UvrA failed to stimulate the UvrB DNA damage-associated ATPase activity. Electrophoretic mobility shift analysis was used to monitor UvrB loading onto damaged DNA with wild-type UvrA or ZnG UvrA. The ZnG UvrA protein showed a 30-60% reduction in UvrB loading as compared with the amount of UvrB loaded by wild-type UvrA. These data demonstrate that the C-terminal zinc finger of UvrA is required for regulation of damage-specific DNA binding.     

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.     

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.     

Sequences of the E. coli uvrB gene and protein.

The UvrB protein is one of the three subunits of the E. coli ABC excinuclease. We have reported the sequences of the other two subunits, the UvrA and UvrC proteins. In this paper the sequence of the UvrB protein is presented. The protein sequence was determined from the DNA sequence of the uvrB gene and was confirmed by sequencing the NH2-terminus of the UvrB protein and analyzing its overall amino acid composition. The coding region of uvrB is 2019 basepairs, specifying a protein of 672 amino acids and Mr of 76,118. The sequence of the UvrB protein shows a moderate level of homology to that of the UvrC protein and to the ATP binding site of the UvrA protein. During purification of UvrB protein a proteolytic product, UvrB, is produced in high quantities. We find that UvrB results from removal of about 40 amino acids from the COOH-terminus of the UvrB protein. The uvrB gene has complex regulatory features. On the 5' side, the coding region is preceded by 3 promoters, a DnaA box and an SOS box. On the 3' side the gene is followed by an REP (Repetitive Extragenic Palindrome) sequence which has been implicated in gene regulation by an unknown mechanism.     

The nucleotide excision repair protein UvrB, a helicase-like enzyme with a catch

Nucleotide excision repair (NER) is a universal DNA repair mechanism found in all three kingdoms of life. Its ability to repair a broad range of DNA lesions sets NER apart from other repair mechanisms. NER systems recognize the damaged DNA strand and cleave it 3', then 5' to the lesion. After the oligonucleotide containing the lesion is removed, repair synthesis fills the resulting gap. UvrB is the central component of bacterial NER. It is directly involved in distinguishing damaged from undamaged DNA and guides the DNA from recognition to repair synthesis. Recently solved structures of UvrB from different organisms represent the first high-resolution view into bacterial NER. The structures provide detailed insight into the domain architecture of UvrB and, through comparison, suggest possible domain movements. The structure of UvrB consists of five domains. Domains 1a and 3 bind ATP at the inter-domain interface and share high structural similarity to helicases of superfamilies I and II. Not related to helicase structures, domains 2 and 4 are involved in interactions with either UvrA or UvrC, whereas domain 1b was implicated for DNA binding. The structures indicate that ATP binding and hydrolysis is associated with domain motions. UvrB's ATPase activity, however, is not coupled to the separation of long DNA duplexes as in helicases, but rather leads to the formation of the preincision complex with the damaged DNA substrate. The location of conserved residues and structural comparisons with helicase-DNA structures suggest how UvrB might bind to DNA. A model of the UvrB-DNA interaction in which a beta-hairpin of UvrB inserts between the DNA double strand has been proposed recently. This padlock model is developed further to suggest two distinct consequences of domain motion: in the UvrA(2)B-DNA complex, domain motions lead to translocation along the DNA, whereas in the tight UvrB-DNA pre-incision complex, they lead to distortion of the 3' incision site.     

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.     

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.     

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.     

ATPase activity of the UvrA and UvrAB protein complexes of the Escherichia coli UvrABC endonuclease.

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.     

Crystal structure of Bacillus stearothermophilus UvrA provides insight into ATP-modulated dimerization, UvrB interaction, and DNA binding

The nucleotide excision repair pathway corrects many structurally unrelated DNA lesions. Damage recognition in bacteria is performed by UvrA, a member of the ABC ATPase superfamily whose functional form is a dimer with four nucleotide-binding domains (NBDs), two per protomer. In the 3.2 A structure of UvrA from Bacillus stearothermophilus, we observe that the nucleotide-binding sites are formed in an intramolecular fashion and are not at the dimer interface as is typically found in other ABC ATPases. UvrA also harbors two unique domains; we show that one of these is required for interaction with UvrB, its partner in lesion recognition. In addition, UvrA contains three zinc modules, the number and ligand sphere of which differ from previously published models. Structural analysis, biochemical experiments, surface electrostatics, and sequence conservation form the basis for models of ATP-modulated dimerization, UvrA-UvrB interaction, and DNA binding during the search for lesions.     

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.