Uvr excision repair protein complex of Escherichia coli binds to the convex side of a cisplatin-induced kink in the DNA.

The Escherichia coli UvrABC endonuclease is capable of initiating the repair of a wide variety of DNA damages. To study the binding of the UvrAB complex to the DNA at the site of a lesion we have constructed a synthetic DNA fragment with a defined cis-diamminedichloroplatinum(II) (cis-Pt).GG adduct. The cis-Pt.GG is the major adduct after treatment of DNA with the antitumor agent cisplatin. Binding to the DNA at the site of the defined lesion was studied with DNase I and MPE.Fe(II) hydroxyl radical footprinting. The results indicate that the UvrAB complex binds to the convex side of the kink in the DNA caused by the cis-Pt.GG adduct. Concerted incisions of the damaged strand by the UvrABC endonuclease were at the 8th phosphodiester bond 5' to and at the 4th bond 3' of the adjacent guanines. An additional incision was found at the 15th phosphodiester bond 5' to the damaged site. This extra incision was stimulated by a high concentration of UvrC.     

The repair of psoralen monoadducts by the Escherichia coli UvrABC endonuclease.

We have examined the interactions of UvrABC endonuclease with DNA containing the monoadducts of 8-methoxypsoralen (8-MOP) and 4,5',8-trimethylpsoralen (TMP). The UvrA and UvrB proteins were found to form a stable complex on DNA that contains the psoralen monoadducts. Subsequent binding of UvrC protein to this complex activates the UvrABC endonuclease activity. As in the case of incision at pyrimidine dimers, a stable protein-DNA complex was observed after the incision events. For both 8-MOP and TMP, the UvrABC endonuclease incised the monoadduct-containing strand of DNA on the two sides of the monoadduct with 12 bases included between the two cuts. One incision was at the 8th phosphodiester bond on the 5' side of the modified base. The other incision was at the 5th phosphodiester bond 3' to the modified base. The UvrABC endonuclease incision data revealed that the reactivity of psoralens is 5'TpA greater than 5'ApT greater than 5'TpG.     

UvrAB activity at a damaged DNA site: is unpaired DNA present?

To study the activity of the Escherichia coli UvrA and UvrB nucleotide excision repair proteins during the formation of the pre-incision complex at a damaged DNA site, we used substrates with modifications around a single 2-(acetylamino)fluorene (AAF) lesion. Based on the release of AAF-containing oligonucleotides from a single-stranded DNA circle, we conclude that during interaction with our substrates UvrAB introduces changes in DNA which are localized at the lesion and are limited to 1-3 bp. Since these changes might include a denaturation of DNA at the lesion site and, consequently, a bubble structure might be present in a pre-incision complex, we studied incision activity of UvrABC excinuclease on substrates with 1-4 unpaired bases next to an AAF adduct. Opening more than one base on either or both sides of the lesion caused a significant decrease in the incision activity of UvrABC, but did not change the position of the incision sites. We conclude that the UvrAB action leading to a pre-incision complex does not include the formation of a bubble intermediate generated by extensive denaturation of base pairs.     

DNA base composition determines the specificity of UvrABC endonuclease incision of a psoralen cross-link

The sequences flanking a psoralen interstrand cross-link may determine how it is repaired. Our comparison of the Escherichia coli UvrABC endonuclease incision of a variety of specific cross-link sequences in a single natural DNA fragment showed that DNA base composition determines which of two cross-linked DNA strands will be incised. G/C enrichment of the region 6-12 bases 5' of the modified T on the furan-side strand results in preferential incision of the furan-side strand. When the G/C-rich region is on the 3' side, or on neither side, incisions occur on either strand. These effects of DNA base composition suggest that UvrAB can bind in two ways to a psoralen cross-link.     

UvrABC incision of N-methylmitomycin A-DNA monoadducts and cross-links

The Escherichia coli UvrABC endonuclease is a multisubunit enzyme that initiates the repair of a wide variety of DNA lesions in vivo by making dual incisions on a damaged strand at the eighth or ninth phosphodiester bond 5' and the fourth or fifth phosphodiester bond 3' to the modified base. It has been hypothesized that UvrABC is able to recognize a broad spectrum of lesions because it does not recognize the lesion per se but rather gross helical distortions that the lesion induces in the DNA. Several lesions have recently been studied which are thermal stabilizing and are not believed to distort the DNA grossly, including the CC-1065-N-3-adenine and anthramycin-N-2-guanine adducts. We have studied the activity of UvrABC in vitro on another thermal stabilizing and nondistortive adduct, N-methylmitomycin A (NMA), a bifunctional DNA-alkylating agent that reacts with guanine on the side facing the minor groove, yielding either monoadducts or interstrand cross-links. NMA adducts increase the thermal stability of DNA, and theoretical calculations indicate that NMA adducts do not grossly distort the DNA helix. Our results show that UvrABC makes incisions at the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to an NMA monoadduct, consistent with the incision pattern observed for the majority of other lesions that are also recognized by UvrABC. DNA containing a site-specific NMA cross-link was also recognized and incised by UvrABC. The rate of incision of NMA cross-linked DNA was about 200-fold higher in supercoiled molecules than in relaxed molecules, whereas the rate of incision of DNA containing NMA monoadducts was stimulated approximately 2-fold by supercoiling. The signal for UvrABC recognition and incision of damaged DNA is discussed in relation to the ability of UvrABC to incise NMA adducts as well as other nondistortive lesions.     

A specific 3'' exonuclease activity of UvrABC.

Specific cutting of undamaged DNA by UvrABC nuclease is observed. It occurs seven nucleotides (nt) from the 3' terminus of oligonucleotides annealed to single-stranded M13 DNA circles. Although the location of the UvrABC cut on undamaged DNA is similar to that of the cut on the 5' side of a damaged DNA site during the dual incision reaction, the cut of undamaged DNA is not an intermediate in the dual incision step. On DNA duplexes with a single AAF adduct, the anticipated cut at the eighth phosphodiester bond 5' of the lesion is present, but extra cuts at 7-nt increments are observed at the 15th and 22nd phosphodiester bonds. We suggest that these additional cuts are made by the UvrABC activity observed on undamaged DNA; such activity is referred to as ABC 3' exonuclease and may play a significant role by providing a suitable gap for RecA-mediated recombinational exchanges during repair of interstrand crosslinks and closely opposed lesions. This ABC 3' exonuclease activity depends on higher concentrations of Uvr proteins as compared with dual incision and may be relevant to reactions that occur when UvrA and UvrB are increased during SOS induction.     

Analysis of UvrABC endonuclease reaction intermediates on cisplatin-damaged DNA using mobility shift gel electrophoresis.

One of the least understood steps in the UvrABC mediated excision repair process is the recognition of lesions in the DNA. The isolation of different reaction intermediates is of vital importance for the unraveling of the mechanism. A mobility shift gel electrophoresis assay is described which visualizes such intermediates. After incubation of a DNA substrate containing a specific cisplatin adduct with UvrA alone or with UvrA and UvrB, UvrA.DNA, UvrAB.DNA and UvrB.DNA complexes were observed which could be identified using specific antibodies. At low UvrA concentrations in the presence of UvrB only the UvrB.DNA complex is observed. Bands corresponding to the UvrAB.DNA complex and also other nonspecific bands are found at relatively high UvrA concentrations. The DNase-I footprint for the UvrAB.- and UvrB.DNA complex are very similar and protect about 20 bases. Both complexes are incised in the presence of UvrC with comparable efficiency. The UvrAB.- and the UvrB.DNA complex were both incised at the 8th phosphodiester bond 5' to a specific cisplatin adduct. In addition the UvrAB.DNA complex could also be incised at the 15th phosphodiesterbond 5' to the damaged site. The results suggest that the UvrB.DNA complex is the natural substrate for UvrC-induced incision.     

Incision of damaged versus nondamaged DNA by the Escherichia coli UvrABC proteins.

Incision of damaged DNA by the Escherichia coli UvrABC endonuclease requires the UvrA, UvrB, and UvrC proteins as well as ATP hydrolysis. This incision reaction can be divided into three steps: site recognition, preincision complex formation, and incision. UvrAB is able to execute the first two steps in the reaction while the addition of UvrC is required for the incision of DNA. This incision reaction does not require ATP hydrolysis and results in the formation of a tight UvrABC post-incision complex and the generation of an oligomer of approximately 12 nucleotides. At high UvrABC concentrations the specificity of the incision for damaged DNA is decreased and significant incision of undamaged DNA occurs. Analogous to damage specific incision, this type of incision leads to generation of an oligonucleotide, but in this case the size is approximately 9 nucleotides in length. Further evidence shows that the combination of UvrB and UvrC proteins can generate a significant amount of a similar size product on undamaged DNA. In addition, the UvrC protein alone can generate a small amount of the same product. Immunological characterization of the weak nuclease activity seen with UvrC indicates that the activity is very tightly associated with the purified UvrC protein.     

The limited strand-separating activity of the UvrAB protein complex and its role in the recognition of DNA damage.

The recognition by Escherichia coli Uvr nucleotide excision repair proteins of a variety of lesions with diverse chemical structures and the presence of helicase activity in the UvrAB complex which can displace short oligonucleotides annealed to single-stranded DNA led to a model in which this activity moves UvrAB along undamaged DNA to damaged sites where the lesion blocks further translocation and the protein-DNA pre-incision complex is formed. To evaluate this mechanism for damage recognition, we constructed substrates with oligonucleotides of different lengths annealed to single-stranded DNA circles and placed a single 2-(acetylamino)fluorene (AAF) lesion either on the oligonucleotide or on the circle. For the substrates with no lesion, the UvrAB complex effectively displaced a 22-mer but not a 27-mer or longer fragments. The presence of AAF on the oligonucleotide significantly increased the release of the 27-mer but oligomers of 30 or longer were not separated. Placing the lesion on the circular strand did not block the release of the fragments. Instead, the releasing activity of UvrAB was stimulated and also depended on the length of the annealed oligonucleotide. These observations do not agree with the predictions of a damage recognition mechanism that depends on helicase-driven translocation. Most likely, the strand-separating activity of UvrAB is a consequence of local changes occurring during the formation of a DNA-protein pre-incision complex at the damaged site and is not due to translocation of the protein along undamaged DNA to locate a lesion.     

Involvement of a cryptic ATPase activity of UvrB and its proteolysis product, UvrB* in DNA repair.

The incision of damaged DNA by the Escherichia coli UvrABC endonuclease requires ATP hydrolysis. Although the deduced sequence of the UvrB protein suggests a putative ATP binding site, no nucleoside triphosphatase activity is demonstrable with the purified UvrB protein. The UvrB protein is specifically proteolyzed in E. coli cell extracts to yield a 70 kD fragment, referred to as UvrB*, which has been purified and is shown to possess a single-strand DNA dependent ATPase activity. Substrate specificity and kinetic analyses of UvrB* catalyzed nucleotide hydrolysis indicate that the stimulation in DNA dependent ATPase activity following formation of the UvrAB complex results from the activation of the normally sequestered UvrB associated ATPase. Using nucleotide analogues, it can be shown that this activity is essential to the DNA incision reaction carried out by the UvrABC complex.     

In vitro repair of psoralen-DNA cross-links by RecA, UvrABC, and the 5'-exonuclease of DNA polymerase I

Psoralens produce DNA interstrand cross-links which are thought to be repaired via a sequential excision and recombination mechanism in Escherichia coli. The first round of incision by UvrABC has been characterized: it results in 11-base oligonucleotide cross-linked to an intact DNA strand (Van Houten, B., Gamper, B., Holbrook, S.R., Hearst, J.E., and Sancar, A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8077-8081). In the present work, DNA substrates containing 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) cross-links in defined positions are constructed and used to analyze the other steps in repair. It is shown that RecA protein mediates strand transfer past an oligonucleotide cross-linked to a single-stranded DNA circle and that the resulting heteroduplex is a substrate for the UvrABC complex: it excises a double-stranded oligonucleotide which contains the HMT cross-link. It is also found that the first round of UvrABC incision does not lead directly to strand exchange but that an intervening step is needed. That step is carried out in vitro by the 5'-exonuclease activity of DNA polymerase I (pol I) which creates a single-stranded DNA region (a gap) at an incised cross-link such that RecA can initiate strand exchange. Studies using cross-linked oligonucleotides showed that the gap produced by pol I results from the inability of the polymerase to add nucleotides to a 3'-OH end two to three nucleotides away from the furan side of an HMT cross-link. Pol I can, however, extend a 3'-OH end next to the pyrone side of the cross-link. Since UvrABC incises predominantly the furan side of psoralen cross-links in duplex DNA, this discrepancy has important consequences for repair.     

The UvrABC endonuclease system of Escherichia coli--a view from Baltimore

Nucleotide excision is initiated by the UvrABC endonuclease system in which the initial DNA interaction is with UvrA which was dimerized in the presence of ATP. Nucleoprotein formation most likely takes place on undamaged regions of DNA by (UvrA)2 which has been dimerized in the presence of ATP. Topological unwinding of DNA, driven by ATP binding, is increased by the presence of UvrB to approximately a single helical turn. The Uvr(A)2B complex translocates to a damaged site by the combined Uvr(A)2B helicase in which the driving force is provided by the UvrB-associated ATPase. The dual incision reaction is initiated by the binding of the UvrC protein to the Uvr(A)2B-nucleoprotein complex. The proteins in this post-incision nucleoprotein complex do not turn over and require the presence of the UvrD protein and DNA polymerase I under polymerizing conditions. The final integrity of the DNA strands is restored with polynucleotide ligase.     

Involvement of a cryptic ATPase activity of UvrB and its proteolysis product, UvrB* in DNA repair

The incision of damaged DNA by the Escherichia coli UvrABC endonuclease requires ATP hydrolysis. Although the deduced sequence of the UvrB protein suggests a putative ATP binding site, no nucleoside triphosphatase activity is demonstrable with the purified UvrB protein. The UvrB protein is specifically proteolyzed in E. coli cell extracts to yield a 70 kD fragment, referred to as UvrB*, which has been purified and is shown to possess a single-strand DNA dependent ATPase activity. Substrate specificity and kinetic analyses of UvrB* catalyzed nucleotide hydrolysis indicate that the stimulation in DNA dependent ATPase activity following formation of the UvrAB complex results from the activation of the normally sequestered UvrB associated ATPase. Using nucleotide analogues, it can be shown that this activity is essential to the DNA incision reaction carried out by the UvrABC complex.     

Initiation of the UvrABC nuclease cleavage reaction. Efficiency of incision is not correlated with UvrA binding affinity

The incision steps of Escherichia coli nucleotide excision repair are mediated by the UvrABC nuclease complex. We have previously shown that the UvrABC nuclease specifically incises apyrimidinic (AP) sites less efficiently than o-benzylhydroxylamine-modified apyrimidinic (BA) sites. To investigate these differences, quantitative DNase I footprinting titration studies were performed. The UvrA binding isotherms were similar for both the AP site (Kd = 6 x 10(-9) M) and the bulkier BA lesion (Kd = 14 x 10(-9) M), despite the fact that the extent of incision differs for these two lesions. It was also found that the relative binding affinity of the preincision UvrA2B complex to the AP and BA substrates differs significantly with estimated apparent equilibrium dissociation constants (Kd) of 4 x 10(-9) M and 80 x 10(-9) to 120 x 10(-9) M, respectively. These results indicate that incision efficiency does not correlate to UvrA binding affinity, but is a direct result of interactions between the UvrA2B complex and the site of the DNA damage. It is also shown that high UvrA concentrations are inhibitory to the UvrABC nuclease reaction.     

Robust incision of Benoz[a]pyrene-7,8-dihyrodiol-9,10-epoxide-DNA adducts by a recombinant thermoresistant interspecies combination UvrABC endonuclease system

Prokaryotic DNA repair nucleases are useful reagents for detecting DNA lesions. UvrABC endonuclease, encoded by the UvrA, UvrB, and UvrC genes can incise DNA containing bulky nucleotide adducts and intrastrand cross-links. UvrA, UvrB, and UvrC were cloned from Bacillus caldotenax (Bca)and UvrC from Thermatoga maritima (Tma), and recombinant proteins were overexpressed in and purified from Escherichia coli. Incision activities of UvrABC composed of all Bca-derived subunits (UvrABC(Bca)) and an interspecies combination UvrABC composed of Bca-derived UvrA and UvrB and Tma-derived UvrC (UvrABC(Tma)) were compared on benoz[a]pyrene-7,8-dihyrodiol-9,10-epoxide (BPDE)-adducted substrates. Both UvrABC(Bca) and UvrABC(Tma) specifically incised both BPDE-adducted plasmid DNAs and site-specifically modified 50-bp oligonucleotides containing a single (+)-trans- or (+)-cis-BPDE adduct. Incision activity was maximal at 55-60 degrees C. However, UvrABC(Tma) was more robust than UvrABC(Bca) with 4-fold greater incision activity on BPDE-adducted oligonucleotides and 1.5-fold greater on [(3)H]BPDE-adducted plasmid DNAs. Remarkably, UvrABC(Bca) incised only at the eighth phosphodiester bond 5' to the BPDE-modified guanosine. In contrast, UvrABC(Tma) performed dual incision, cutting at both the fifth phosphodiester bond 3' and eighth phosphodiester bond 5' from BPDE-modified guanosine. BPDE adduct stereochemistry influenced incision activity, and cis adducts on oligonucleotide substrates were incised more efficiently than trans adducts by both UvrABC(Bca) and UvrABC(Tma). UvrAB-DNA complex formation was similar with (+)-trans- and (+)-cis-BPDE-adducted substrates, suggesting that UvrAB binds both adducts equally and that adduct configuration modifies UvrC recognition of the UvrAB-DNA complex. The dual incision capabilities and higher incision activity of UvrABC(Tma) make it a robust tool for DNA adduct studies.     

Damage repertoire of the Escherichia coli UvrABC nuclease complex includes abasic sites, base-damage analogues, and lesions containing adjacent 5' or 3' nicks

Using oligonucleotide synthesis, we demonstrate a rapid and efficient method for the construction of DNA duplexes containing defined DNA lesions at specific positions. These DNA lesions include apyrimidinic sites, reduced apyrimidinic sites, and base-damage analogues consisting of O-methyl- or O-benzylhydroxylamine-modified apyrimidinic sites. A 49 base pair DNA duplex containing these lesions was specifically incised by the UvrABC nuclease complex. The incision sites occurred predominantly at the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to the lesion. Multiple incisions were observed 3' to the lesion. The extent of DNA incisions was base-damage analogues greater than reduced apyrimidinic sites greater than apyrimidinic sites. Introduction of 3' or 5' nicks at the site of a base-damage analogue by treatment of these substrates with either endonuclease III or endonuclease IV reduced, but did not abolish, subsequent incision by the UvrABC complex, whereas introduction of a 3' nick at an abasic site increased the incision efficiency of the UvrABC complex. These data demonstrate a convergence of base and nucleotide excision repair pathways in the removal of specific base damages.     

Mutagenesis induced by site specifically placed 4''-hydroxymethyl-4,5'',8-trimethylpsoralen adducts.

Closed circular double stranded M13mp19 DNA containing a site-specifically placed HMT (4'-hydroxymethyl-4-5'-8-trimethylpsoralen) monoadduct or crosslink was synthesized in vitro. The damaged DNA were scored for loss of infectivity by transfection into repair proficient or deficient E. coli and into SOS induced E. coli. Mutant phages were detected by the loss of alpha-complementation between the viral and the host Lac Z genes or by the acquisition of resistance to kpn I digestion. Our results indicate that HMT mutagenesis is targeted and that deletion or transversion of the modified thymidine is the predominant sequence change elicited by a monoadduct or a crosslink. Transfection of the monoadducted DNA into a Uvr A deficient strain did not change the mutation pattern but did increase the respective mutation frequencies. Transfection of the crosslinked DNA into a SOS induced host resulted in the appearence of other types of mutations attributable to an increase in both targeted and untargeted mutations.     

Initiation of repair of DNA-polypeptide cross-links by the UvrABC nuclease

Although the biochemical pathways that repair DNA-protein cross-links have not been clearly elucidated, it has been proposed that the partial proteolysis of cross-linked proteins into smaller oligopeptides constitutes an initial step in removal of these lesions by nucleotide excision repair (NER). To test the validity of this repair model, several site-specific DNA-peptide and DNA-protein cross-links were engineered via linkage at (1) an acrolein-derived gamma-hydroxypropanodeoxyguanosine adduct and (2) an apurinic/apyrimidinic site, and the initiation of repair was examined in vitro using recombinant proteins UvrA and UvrB from Bacillus caldotenax and UvrC from Thermotoga maritima. The polypeptides cross-linked to DNA were Lys-Trp-Lys-Lys, Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr, and the 16 kDa protein, T4 pyrimidine dimer glycosylase/apurinic/apyrimidinic site lyase. For the substrates examined, DNA incision required the coordinated action of all three proteins and occurred at the eighth phosphodiester bond 5' to the lesion. The incision rates for DNA-peptide cross-links were comparable to or greater than that measured on fluorescein-adducted DNA, an excellent substrate for UvrABC. Incision rates were dependent on both the site of covalent attachment on the DNA and the size of the bound peptide. Importantly, incision of a DNA-protein cross-link occurred at a rate approximately 3.5-8-fold slower than the rates observed for DNA-peptide cross-links. Thus, direct evidence has been obtained indicating that (1) DNA-peptide cross-links can be efficiently incised by the NER proteins and (2) DNA-peptide cross-links are preferable substrates for this system relative to DNA-protein cross-links. These data suggest that proteolytic degradation of DNA-protein cross-links may be an important processing step in facilitating NER.     

Wavelength dependence for the photoreactions of DNA-psoralen monoadducts. 2. Photo-cross-linking of monoadducts

The photoreactions of HMT [4'-(hydroxymethyl)-4,5',8-trimethylpsoralen] monoadducts in double-stranded DNA have been studied with complementary oligonucleotides. The HMT was first attached to the thymidine residue in the oligonucleotide 5'-GAAGCTACGAGC-3' as either a furan-side monoadduct or a pyrone-side monoadduct. The HMT-monoadducted oligonucleotide was then hybridized to the complementary oligonucleotide 5'-GCTCGTAGCTTC-3' and irradiated with monochromatic light. In the case of the pyrone-side monoadducted oligonucleotide, photoreversal was the predominant reaction, and very little cross-link was formed at all wavelengths. The course of the photoreaction of the double-stranded furan-side monoadducted oligonucleotide was dependent on the irradiation wavelength. At wavelengths below 313 nm, both photoreversal and photo-cross-linking occurred. At wavelengths above 313 nm, photoreversal of the monoadduct could not be detected, and photo-cross-linking occurred efficiently with a quantum yield of 2.4 X 10(-2).     

Characterization of the helicase activity of the Escherichia coli UvrAB protein complex

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.