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.     

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.     

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.     

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.     

ATP binding to two sites is necessary for dimerization of nucleotide-binding domains of ABC proteins

ATP binding cassette (ABC) transporters have a functional unit formed by two transmembrane domains and two nucleotide binding domains (NBDs). ATP-bound NBDs dimerize in a head-to-tail arrangement, with two nucleotides sandwiched at the dimer interface. Both NBDs contribute residues to each of the two nucleotide-binding sites (NBSs) in the dimer. In previous studies, we showed that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii forms ATP-bound dimers that dissociate completely following hydrolysis of one of the two bound ATP molecules. Since hydrolysis of ATP at one NBS is sufficient to drive dimer dissociation, it is unclear why all ABC proteins contain two NBSs. Here, we used luminescence resonance energy transfer (LRET) to study ATP-induced formation of NBD homodimers containing two NBSs competent for ATP binding, and NBD heterodimers with one active NBS and one binding-defective NBS. The results showed that binding of two ATP molecules is necessary for NBD dimerization. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dissociation, but two binding sites are required to form the ATP-sandwich NBD dimer necessary for hydrolysis.     

Modeling the interactions of the nucleotide excision repair UvrA(2) dimer with DNA

The UvrA protein initiates the DNA damage recognition process by the bacterial nucleotide excision repair (NER) system. Recently, crystallographic structures of holo-UvrA(2) dimers from two different microorganisms have been released (Protein Data Bank entries 2r6f , 2vf7 , and 2vf8 ). However, the details of the DNA binding by UvrA(2) and other peculiarities involved in the damage recognition process remain unknown. We have undertaken a molecular modeling approach to appraise the possible modes of DNA-UvrA(2) interaction using molecular docking and short-scale guided molecular dynamics [continuum field, constrained, and/or unrestricted simulated annealing (SA)], taking into account the three-dimensional location of a series of mutation-identified UvrA residues implicated in DNA binding. The molecular docking was based on the assumptions that the UvrA(2) dimer is preformed prior to DNA binding and that no major protein conformational rearrangements, except moderate domain reorientations, are required for binding of undamaged DNA. As a first approximation, DNA was treated as a rigid ligand. From the electrostatic relief of the ventral surface of UvrA(2), we initially identified three, noncollinear DNA binding paths. Each of the three resulting nucleoprotein complexes (C1, C2, and C3) was analyzed separately, including calculation of binding energies, the number and type of interaction residues (including mutated ones), and the predominant mode of translational and rotational motion of specific protein domains after SA to ensure improved DNA binding. The UvrA(2) dimer can accommodate DNA in all three orientations, albeit with different binding strengths. One of the UvrA(2)-DNA complexes (C1) fulfilled most of the requirements (high interaction energy, proximity of DNA to mutated residues, etc.) expected for a natural, high-affinity DNA binding site. This nucleoprotein presents a structural organization that is designed to clamp and bend double-stranded DNA. We examined the binding site in more detail by docking DNAs of significantly different (AT- vs CG-enriched) sequences and by submitting the complexes to DNA-unrestricted SA. It was found that in a manner independent of the DNA sequence and applied MD protocols, UvrA(2) favors binding of a bent and unwound undamaged DNA, with a kink positioned in the proximity of the Zn3 hairpins, anticollinearly aligned at the bottom of the ventral protein surface. It is further hypothesized that the Zn3 modules play an essential role in the damage recognition process and that the apparent existence of a family of DNA binding sites might be biologically relevant. Our data should prove to be useful in rational (structure-based) mutation studies.     

ATP increases the affinity between MutS ATPase domains. Implications for ATP hydrolysis and conformational changes

MutS is the key protein of the Escherichia coli DNA mismatch repair system. It recognizes mispaired and unpaired bases and has intrinsic ATPase activity. ATP binding after mismatch recognition by MutS serves as a switch that enables MutL binding and the subsequent initiation of mismatch repair. However, the mechanism of this switch is poorly understood. We have investigated the effects of ATP binding on the MutS structure. Crystallographic studies of ATP-soaked crystals of MutS show a trapped intermediate, with ATP in the nucleotide-binding site. Local rearrangements of several residues around the nucleotide-binding site suggest a movement of the two ATPase domains of the MutS dimer toward each other. Analytical ultracentrifugation experiments confirm such a rearrangement, showing increased affinity between the ATPase domains upon ATP binding and decreased affinity in the presence of ADP. Mutations of specific residues in the nucleotide-binding domain reduce the dimer affinity of the ATPase domains. In addition, ATP-induced release of DNA is strongly reduced in these mutants, suggesting that the two activities are coupled. Hence, it seems plausible that modulation of the affinity between ATPase domains is the driving force for conformational changes in the MutS dimer. These changes are driven by distinct amino acids in the nucleotide-binding site and form the basis for long-range interactions between the ATPase domains and DNA-binding domains and subsequent binding of MutL and initiation of mismatch repair.     

ATP binding cassette systems: structures, mechanisms, and functions

ATP-binding cassette (ABC) systems are found in all three domains of life and in some giant viruses and form one of the largest protein superfamilies. Most family members are transport proteins that couple the free energy of ATP hydrolysis to the translocation of solutes across a biological membrane. The energizing module is also used to drive non-transport processes associated, e.g., with DNA repair and protein translation. Many ABC proteins are of considerable medical importance. In humans, dysfunction of at least eighteen out of 49 ABC transporters is associated with disease, such as cystic fibrosis, Tangier disease, adrenoleukodystrophy or Stargardt’s macular degeneration. In prokaryotes, ABC proteins confer resistance to antibiotics, secrete virulence factors and envelope components, or mediate the uptake of a large variety of nutrients. Canonical ABC transporters share a common structural organization comprising two transmembrane domains (TMDs) that form the translocation pore and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. In this Mini-Review, we summarize recent structural and biochemical data obtained from both prokaryotic and eukaryotic model systems.     

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.     

Subunit interactions in ABC transporters: towards a functional architecture

The ABC superfamily is a diverse group of integral membrane proteins involved in the ATP-dependent transport of solutes across biological membranes in both prokaryotes and eukaryotes. Although ABC transporters have been studied for over 30 years, very little is known about the mechanism by which the energy of ATP hydrolysis is used to transport substrate across the membrane. The recent report of the high resolution crystal structure of HisP, the nucleotide-binding subunit of the histidine permease complex of Salmonella typhimurium, represents a significant breakthrough toward the elucidation of the mechanism of solute translocation by ABC transporters. In this review, we use data from the crystallographic structures of HisP and other nucleotide-binding proteins, combined with sequence analysis of a subset of atypical ABC transporters, to argue a new model for the dimerisation of the nucleotide-binding domains that embraces the notion that the C motif from one subunit forms part of the ATP-binding site in the opposite subunit. We incorporate this dimerisation of the ATP-binding domains into our recently reported beta-barrel model for P-glycoprotein and present a general model for the cooperative interaction of the two nucleotide-binding domains and the translocation of mechanical energy to the transmembrane domains in ABC transporters.     

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 the different intermediates in the interaction of (A)BC excinuclease with its substrates by DNase I footprinting on two uniquely modified oligonucleotides

(A)BC excinuclease is the enzymatic activity resulting from the joint actions of UvrA, UvrB and UvrC proteins of Escherichia coli. The enzyme removes from DNA many types of adducts of dissimilar structures with different efficiencies. To understand the mechanism of substrate recognition and the basis of enzyme specificity, we investigated the interactions of the three subunits with two synthetic substrates, one containing a psoralen-thymine monoadduct and the other a thymine dimer. Using DNase I as a probe, we found that UvrA makes a 33 base-pair footprint around the psoralen-thymine adduct and that UvrA-UvrB make a 45 base-pair asymmetric footprint characterized by a hypersensitive site 11 nucleotides 5' to the adduct and protection mostly on the 3' side of the damage. Conditions that favor dissociation of UvrA from the UvrA-UvrB-DNA complex, such as addition of excess undamaged DNA to the reaction mixture, resulted in the formation of a 19 base-pair UvrB footprint. In contrast, a thymine dimer in a similar sequence context failed to elicit a UvrA, a UvrA-UvrB or UvrB footprint and gave rise to a relatively weak DNase I hypersensitive site typical of a UvrA-UvrB complex. Dissociation of UvrA from the UvrA-UvrB-DNA complex stimulated the rate of incision of both substrates upon addition of UvrC, leading us to conclude that UvrA is not a part of the incision complex and that it actually interferes with incision. The extent of incision of the two substrates upon addition of UvrC (70% for the psoralen adduct and 20% for the thymine dimer) was proportional to the extent of formation of the UvrA-UvrB-DNA (i.e. UvrB-DNA) complex, indicating that substrate discrimination occurs at the preincision step.     

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.     

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.     

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.     

Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers

The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we used luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation.     

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.     

ABC transporters, mechanisms and biology: an overview

This chapter concentrates mainly on structural and mechanistic aspects of ABC (ATP-binding cassette) transporters and, as an example of the physiological significance of these proteins, on lipid transport, vitally important for human health. The chapter considers those aspects of ABC transporter function that appear reasonably well established, those that remain controversial and what appear to be emerging themes. Although we have seen dramatic progress in ABC protein studies in the last 20 years, we are still far from a detailed molecular understanding of function. Nevertheless two critical steps - capture and release of allocrites (transport substrates) involving a binding cavity in the membrane domain, and hydrolysis of ATP by the NBD (nucleotide-binding domain) dimer - are now described by persuasive and testable models: alternating access, and sequential firing of catalysis sites respectively. However, these need to be tested rigorously by more structural and biochemical studies. Other aspects considered include the level at which ATP binding and dimer activation are controlled, the nature of the power stroke delivering mechanical energy for transport, and some unexpected and intriguing differences between importers and exporters. The chapter also emphasizes that some ABC transporters, although important for elimination of toxic compounds (xenobiotics), are also increasingly seen to play crucial roles in homoeostatic regulation of membrane biogenesis and function through translocation of endogenous allocrites such as cholesterol. Another emerging theme is the identification of accessory domains and partners for ABC proteins, resulting in a corresponding widening of the range of activities. Finally, what are the prospects for translational research and ABC transporters?     

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.     

Recognition and incision of oxidative intrastrand cross-link lesions by UvrABC nuclease

Nucleotide excision repair (NER) is a repair pathway that removes a variety of bulky DNA lesions in both prokaryotic and eukaryotic cells. The perturbation of DNA helix structure caused by the oxidative intrastrand lesions could render them good substrates for the NER pathway. Here we employed Escherichia coli NER enzymes, i.e., UvrA, UvrB, and UvrC, to examine the incision efficiency of duplex DNA carrying three different oxidative intrastrand cross-link lesions, that is, G[8-5]C, G[8-5m]mC, and G[8-5m]T, and two dithymine photoproducts, namely, the cis,syn-cyclobutane pyrimidine dimer (T[c,s]T) and the pyrimidine(6-4)pyrimidone product (T[6-4]T). Our results showed that T[6-4]T was the best substrate for UvrA binding, followed by G[8-5]C, G[8-5m]mC, and G[8-5m]T, and then by T[c,s]T. The efficiencies of the UvrABC incisions of these lesions were consistent with their UvrA binding affinities: the stronger the binding to UvrA, the higher the rate of incision. In addition, flanking DNA sequences appeared to have little effect on the binding affinity of UvrA for G[8-5]C as AG[8-5]CA was only slightly preferred over CG[8-5]CG. Consistently, these two sequences exhibited almost no difference in incision rates. Furthermore, we investigated the thermal stability of dodecameric duplexes containing G[8-5m]mC or G[8-5m]T, and our results revealed that these two lesions destabilized the duplex, due to an increase in the free energy for duplex formation at 37 degrees C, by approximately 5.4 and 3.6 kcal/mol, respectively. The destabilizations to the DNA helix caused by those lesions, for the most part, are correlated with the binding affinities of UvrA and incision rates of UvrABC. Taken together, the results from this study suggest that oxidative intrastrand lesions might be substrates for NER enzymes in vivo.