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.     

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.     

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.     

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.     

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.     

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.     

Controlled degradation by ClpXP protease tunes the levels of the excision repair protein UvrA to the extent of DNA damage

UV irradiation damages DNA and activates expression of genes encoding proteins helpful for survival under DNA stress. These proteins are often deleterious in the absence of DNA damage. Here, we investigate mechanisms used to regulate the levels of DNA-repair proteins during recovery by studying control of the nucleotide excision repair (NER) protein UvrA. We show that UvrA is induced after UV irradiation and reaches maximum levels between ∼20 and 120 min post UV. During post-UV recovery, UvrA levels decrease principally as a result of ClpXP-dependent protein degradation. The rate of UvrA degradation depends on the amount of unrepaired pyrimidine dimers present; this degradation rate is initially slow shortly after UV, but increases as damage is repaired. This increase in UvrA degradation as repair progresses is also influenced by protein–protein interactions. Genetic and in vitro experiments support the conclusion that UvrA–UvrB interactions antagonize degradation. In contrast, Mfd appears to act as an enhancer of UvrA turnover. Thus, our results reveal that a complex network of interactions contribute to tuning the level of UvrA in the cell in response to the extent of DNA damage and nicely mirror findings with excision repair proteins from eukaryotes, which are controlled by proteolysis in a similar manner.     

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.     

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.     

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.     

Investigation of bacterial nucleotide excision repair using single-molecule techniques

Despite three decades of biochemical and structural analysis of the prokaryotic nucleotide excision repair (NER) system, many intriguing questions remain with regard to how the UvrA, UvrB, and UvrC proteins detect, verify and remove a wide range of DNA lesions. Single-molecule techniques have begun to allow more detailed understanding of the kinetics and action mechanism of this complex process. This article reviews how atomic force microscopy and fluorescence microscopy have captured new glimpses of how these proteins work together to mediate NER.     

Cooperative damage recognition by UvrA and UvrB: identification of UvrA residues that mediate DNA binding

Nucleotide excision repair (NER) is responsible for the recognition and removal of numerous structurally unrelated DNA lesions. In prokaryotes, the proteins UvrA, UvrB and UvrC orchestrate the recognition and excision of aberrant lesions from DNA. Despite the progress we have made in understanding the NER pathway, it remains unclear how the UvrA dimer interacts with DNA to facilitate DNA damage recognition. The purpose of this study was to define amino acid residues in UvrA that provide binding energy to DNA. Based on conservation among approximately 300 UvrA sequences and 3D-modeling, two positively charged residues, Lys680 and Arg691, were predicted to be important for DNA binding. Mutagenesis and biochemical analysis of Bacillus caldontenax UvrA variant proteins containing site directed mutations at these residues demonstrate that Lys680 and Arg691 make a significant contribution toward the DNA binding affinity of UvrA. Replacing these side chains with alanine or negatively charged residues decreased UvrA binding 3-37-fold. Survival studies indicated that these mutant proteins complemented a WP2 uvrA(-) strain of bacteria 10-100% of WT UvrA levels. Further analysis by DNase I footprinting of the double UvrA mutant revealed that the UvrA DNA binding defects caused a slower rate of transfer of DNA to UvrB. Consequently, the mutants initiated the oligonucleotide incision assay nearly as well as WT UvrA thus explaining the observed mild phenotype in the survival assay. Based on our findings we propose a model of how UvrA binds to DNA.     

Structure of UvrABC excinuclease-UV-damaged DNA complexes studied by flow linear dichroism. DNA curved by UvrB and UvrC.

The interaction between UvrABC excinuclease from Escherichia coli and ultraviolet light-(UV) damaged DNA was studied by flow linear dichroism. The dichroism signal from DNA was drastically decreased in intensity upon incubation with UvrA and UvrB or whole enzyme in the presence of effector ATP. The change was specific for UV-damaged DNA, and a concluded suppressed DNA orientation suggests the wrapping of DNA around the protein. The incubation with the UvrC subunit alone also somewhat reduces the signal, however, in this case the change was smaller and not specific for UV-damaged DNA. The structural modification of DNA, promoted by the (UvrA2-UvrB) complex, probably facilitates or stabilizes the interaction of the UvrC subunit with DNA for the excision.     

Reduced sulfhydryls maintain specific incision of BPDE-DNA adducts by recombinant thermoresistant Bacillus caldotenax UvrABC endonuclease

Prokaryotic DNA repair nucleases are useful reagents for detecting DNA lesions. Escherichia coli UvrABC endonuclease can incise DNA containing UV photoproducts and bulky chemical adducts. The limited stability of the E. coli UvrABC subunits leads to difficulty in estimating incision efficiency and quantitative adduct detection. To develop a more stable enzyme with greater utility for the detection of DNA adducts, thermoresistant UvrABC endonuclease was cloned from the eubacterium Bacillus caldotenax (Bca) and individual recombinant protein subunits were overexpressed in and purified from E. coli. Here, we show that Bca UvrC that had lost activity or specificity could be restored by dialysis against buffer containing 500 mM KCl and 20mM dithiothreitol. Our data indicate that UvrC solubility depended on high salt concentrations and UvrC nuclease activity and the specificity of incisions depended on the presence of reduced sulfhydryls. Optimal conditions for BCA UvrABC-specific cleavage of plasmid DNAs treated with [3H](+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) (1-5 lesions/plasmid) were developed. Preincubation of substrates with UvrA and UvrB enhanced incision efficiency on damaged substrates and decreased non-specific nuclease activity on undamaged substrates. Under optimal conditions for damaged plasmid incision, approximately 70% of adducts were incised in 1 nM plasmid DNA (2 BPDE adducts/5.4 kbp plasmid) with UvrA at 2.5 nM, UvrB at 62.5 nM, and UvrC at 25 nM. These results demonstrate the potential usefulness of the Bca UvrABC for monitoring the distribution of chemical carcinogen-induced lesions in DNA.     

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.     

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.     

Movement of the β-hairpin in the third zinc-binding module of UvrA is required for DNA damage recognition

Nucleotide excision repair (NER) is distinguished from other DNA repair pathways by its ability to process various DNA lesions. In bacterial NER, UvrA is the key protein that detects damage and initiates the downstream NER cascade. Although it is known that UvrA preferentially binds to damaged DNA, the mechanism for damage recognition is unclear. A β-hairpin in the third Zn-binding module (Zn3hp) of UvrA has been suggested to undergo a conformational change upon DNA binding, and proposed to be important for damage sensing. Here, we investigate the contribution of the dynamics in the Zn3hp structural element to various activities of UvrA during the early steps of NER. By restricting the movement of the Zn3hp using disulfide crosslinking, we showed that the movement of the Zn3hp is required for damage-specific binding, UvrB loading and ATPase activities of UvrA. We individually inactivated each of the nucleotide binding sites in UvrA to investigate its role in the movement of the Zn3hp. Our results suggest that the conformational change of the Zn3hp is controlled by ATP hydrolysis at the distal nucleotide binding site. We propose a bi-phasic damage inspection model of UvrA in which movement of the Zn3hp plays a key role in damage recognition.     

Amplification and purification of UvrA, UvrB, and UvrC proteins of Escherichia coli

The UvrA, UvrB, and UvrC proteins of Escherichia coli are subunits of a DNA repair enzyme, ABC exci nuclease. In order to amplify these proteins, we have joined the artificial canonical promoter tac (Amann E., Brosius, J., and Ptashne, M. (1983) Gene (Amst.) 25, 167-178) to the uvr genes to obtain plasmids that express these genes under the control of the lac repressor. When cells carrying the tac-uvr plasmids are induced by the gratuitous lac inducer isopropyl-beta-D-galactoside the Uvr proteins are overproduced reaching a level of 10-20% of total cellular proteins after 6-8 h of induction. We have developed methods to purify all three Uvr proteins, UvrA, UvrB, and UvrC, in milligram quantities and to near homogeneity from these overproducing cells. The purified UvrA protein is an ATPase but UvrB and UvrC proteins are not. However, UvrB protein stimulates the ATPase activity of UvrA protein by a factor of 1.5 in the presence of double-stranded DNA and by a factor of about 2.6 in the presence of UV-irradiated DNA but not in the absence of DNA.