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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.

The Escherichia coli Uvr(A)BC endonuclease (Uvr(A)BC) initiates nucleotide excision repair of a large variety of DNA damages. The damage recognition and incision steps by the Uvr(A)BC is a complex process utilizing an ATP-dependent DNA helix-tracking activity associated with the UvrA2B1 complex. The latter activity leads to the generation of highly positively supercoiled DNA in the presence of E. coli topoisomerase I in vitro. Such highly positively supercoiled DNA, containing ultraviolet irradiation-induced photoproducts (uvDNA), is resistant to the incision by Uvr(A)BC, whereas the negatively supercoiled and relaxed forms of the uvDNA are effectively incised. The E. coli gyrase can contribute to the above reaction by abolishing the accumulation of highly positively supercoiled uvDNA thereby restoring Uvr(A)BC-catalyzed incision. Eukaryotic (calf thymus) topoisomerase I is able to substitute for gyrase in restoring this Uvr(A)BC-mediated incision reaction. The inability of Uvr(A)BC to incise highly positively supercoiled uvDNA results from the failure of the formation of UvrAB-dependent obligatory intermediates associated with the DNA conformational change. In contrast to Uvr(A)BC, the Micrococcus luteus UV endonuclease efficiently incises uvDNA regardless of its topological state. The in vitro topodynamic system proposed in this study may provide a simple model for studying a topological aspect of nucleotide excision repair and its interaction with other DNA topology-related processes in E. coli.

Based on the binding of the UvrAB complex to a promoter region in transcription open complexes (Ahn, B., and Grossman, L. (1996) J. Biol. Chem. 271, 21453-21461) and the requirement of a single-stranded region for UvrAB helicase activity, we examined the binding of UvrAB proteins to synthetic bubble or loop regions in duplex DNA and the role of these regions in translocation of the UvrAB complex as well as incision of DNA damage. We found that the UvrAB complex was able to bind to bubble and loop regions with an affinity similar to that for damaged DNA in the absence of RNAP. The preferential recognition and incision of damaged sites by the UvrAB complex was observed downstream of the bubble or loop region in the strand complementary to the strand along which the UvrAB complex translocates. These results imply that the bubble region generated in duplex DNA by RNAP serves as a preferred entry site for the translocation of the UvrAB complex, and that preferential binding and unidirectional translocation of the UvrAB complex predetermine where incision is to occur.

The Escherichia coli Uvr(A)BC endonuclease acts in a progression of several distinct steps accompanied by changes in the conformation of macromolecular constituents, the overall architecture of the complex, and its stoichiometry. In order to probe these structural changes, we generated monoclonal antibodies (mAbs) to Uvr proteins. The anti-UvrA mAb, A2D1, recognizing the N-terminal zinc-finger region of UvrA, and the anti-UvrB mAb, B2E2, having an epitope within the 43 C-terminal amino acids of UvrB, were purified and further characterized. It was found that A2D1 mAb interacts in solution both with UvrA-UvrB and UvrA-DNA complexes in the presence of the requisite ATP. This implies that the N-terminal zinc-finger of UvrA doesn't play a direct role in its interactions with UvrB and DNA. On the other hand, A2D1 does inhibit formation of the UvrB-damaged DNA preincision complex, apparently by preventing UvrB delivery by UvrA. The interaction of B2E2 with UvrA-UvrB and nucleoprotein complexes, including UvrB, suggests that the highly hydrophobic C-terminal domain of UvrB (i) doesn't participate in its interaction with UvrA, (ii) is accessible to this mAb in an intermediate UvrA-UvrB DNA helix-tracking complex, and (iii) seems to be directly involved in the formation of the preincision complex. These conclusions are supported by the finding that the neutralizing effect of A2D1 and B2E2 on the Uvr(A)BC endonuclease is significantly decreased if the preincision complex is preformed prior to mAbs addition.

The cysteine 763 residue in the C-terminal zinc-finger region of Escherichia coli UvrA protein was subjected to random mutagenesis, and the results suggested that the UvrA mutants with a small amino acid (Ser, Ala, or Gly) substituting for the cysteine 763 were almost as active as the wild-type in supporting nucleotide excision repair, but its replacement with a large, bulky amino acid (Tyr, Trp, or Phe) rendered the mutants inactive. The C763F mutant UvrA protein was purified for further characterization, and it was found this mutant UvrA protein lost its DNA binding (single-stranded or double-stranded DNA) activity and those other activities dependent on DNA binding, such as formation of damage-specific UvrA2B complexes and the supercoiling reaction. However, this mutant protein retained vigorous ATPase activity and was capable of negatively complementing the wild-type UvrA in JM109 strain. The purified C763F mutant UvrA protein contains a single zinc ion/molecule, half that of the wild-type. It appears that the C763F mutation destabilizes the zinc-anchored structure in the C-terminal zinc finger region, and as a result, the C763F mutant UvrA protein lost its ability to bind DNA.

The biochemical properties of the Escherichia coli UvrA tandem ATPase site mutants in nucleotide excision repair have been studied. In these and earlier studies it was found that ATP binding is required for protein-protein and nucleoprotein association reactions, whereas the dissociation reactions are driven by the hydrolysis of ATP. The self-association of UvrA to form the reactive dimeric species UvrA2 is driven by nucleotide binding, but its dissociation from DNA requires ATP hydrolysis. Similarly, ATP binding drives those allosteric changes in DNA topology during UvrA2-nucleoprotein formation (Oh, E.Y., and Grossman, L. (1986) Nucleic Acids Res. 14, 8557-8571). The manifestation of the UvrB-associated cryptic ATPase requires UvrA and DNA in a helicase-catalyzed supercoiling reaction. The UvrA2B helicase activity requires ATP hydrolysis by the C-terminal ATPase site of UvrA in addition to UvrB. ATP hydrolysis by the C-terminal ATPase site of UvrA also participates in the localization of damaged sites contributing to the formation of damage-specific high affinity nucleoprotein complexes. The levels of complementation to UV survival by the ATPase site mutants of UvrA (Thiagalingam, S., and Grossman, L. (1991) J. Biol. Chem. 266, 11395-11403) correspond to its ability to self-bind and translocate in combination with the UvrB subunit in its search for damaged sites during the preincision mode of nucleotide excision.

The repair by UvrABC endonuclease of two major adducts formed by aflatoxin B1 in DNA was found to be similar. Aflatoxin epoxide was used to generate the aflatoxin B1.N7-guanine adduct which can convert to aflatoxin B1-formamidopyrimidine adduct. The reaction of the aflatoxin B1 epoxide with DNA follows pseudo-first order kinetics. The DNA sequence-specific relative reactivity of the epoxide is the same as previously observed for aflatoxin B1 activated by liver microsomes, therefore strongly reinforcing the notion that aflatoxin B1 reacts with DNA through the epoxide intermediate. For the majority of lesion sites, a high affinity protein-DNA complex was formed from the UvrA and the UvrB proteins with similar efficiency to both adducts, and to pyrimidine dimers, and then nicks the DNA when UvrC was added. The two incisions are at the eighth phosphodiester moiety 5' and the sixth phosphodiester moiety 3' of a modified guanine nucleotide. Both incisions appeared to be concerted. For some sites, the DNA sequence can alter the relative incision efficiency up to 15-fold. However, the majority of these AFB1 lesion structures in most DNA sequences are similar with respect to recognition by this nucleotide excision repair enzyme. Therefore the observation that the aflatoxin B1.N7-guanine lesion is removed rapidly, while the aflatoxin B1-formamidopyrimidine lesion persists in the mammalian cell may have other mechanistic explanations.

The functions of each of the three subunits of the damage-specific UvrABC endonuclease is currently being studied by systematically mutagenizing the corresponding genes to generate mutant proteins for characterization in vitro. In this communication, we describe the construction of C-terminal deletion mutants of the UvrA protein and a procedure to purify the mutant and wild-type UvrA proteins from inclusion bodies in cells overexpressing the recombinant proteins. The method yields highly purified proteins with between 10 and 50% of the specific activity of wild-type UvrA purified by conventional techniques from the soluble fraction. The wild-type UvrA protein purified by this method had the properties of significant and selective loss of activity in assays of incision of damaged DNA, while still retaining high levels of the other unique molecular phenotypic properties associated with intact UvrA. Furthermore, the demonstration of the absolute requirement for zinc during refolding for recovery of activity is the first evidence that the zinc previously shown to be associated with the UvrA protein is in fact a necessary component for its function.

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.

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

The role of UvrB in determining the nucleotide dependence of Escherichia coli excision repair has been investigated. The mutation of lysine 45 in the ATPase motif of UvrB to alanine leads to an acute defect in ATP hydrolysis and failure to support incision of UV-damaged DNA. This ATP hydrolysis activity is not required for interaction of UvrB with UvrA in solution, or for formation of a damage-independent nucleoprotein complex in the presence of UvrA and nucleotide. This UvrB mutant fails, however, to support damage-specific nucleoprotein complex formation, and does not participate in a UvrA-UvrB-dependent helicase-like activity. We conclude from these results that mutation at lysine 45 in the ATPase motif of UvrB specifically inhibits a key step in nucleotide excision repair involving the UvrB ATPase-dependent translocation of nucleoprotein complexes from undamaged to damaged DNA sites.

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

The mechanism of action of the homogeneous preparation of human placental apurinic/apyrimidinic (AP) endonuclease, described in the previous paper (Shaper, N. L., Grafstrom, R. H., and Grossman, L. (1982) J. Biol. Chem. 257, 13455-13458), has been investigated in detail. This enzyme cleaves apyrimidinic DNA both 5' and 3' to the site of damage in a ratio of 60:40, respectively. Even though this enzyme can cleave on both sides of an internal AP site, it does not release deoxyribose 5-phosphate from terminal AP sites. However, a compound, tentatively identified as alpha, beta unsaturated deoxyribose 5-phosphate, is nonenzymatically released only from 5'-terminal AP sites, presumably by a beta-elimination mechanism.

An endonuclease from human placenta has been purified to apparent homogeneity, which acts specifically on DNA containing either apurinic or apyrimidinic sites. The isolation procedure, which results in a 20,000-fold purification and an overall yield of 15%, employs chromatography on a gel of octyl succinic anhydride coupled to agarose by diaminohexane spacers, isoelectric focusing, Sephadex G-75 chromatography, and DNA agarose affinity chromatography. Under conditions in which proteolysis is minimized, this enzyme appears to be the major species of apurinic/apyrimidinic endonuclease. The endonuclease is a monomeric protein with an apparent Mr = 37,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pI of 7.4-7.6, requires Mg2+, is partially stimulated by Mn2+, and is inhibited by EDTA. It has no detectable exonuclease or phosphomonoesterase activity.