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.     

Repair of DNA-containing pyrimidine dimers

Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.     

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.     

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.     

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.     

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.     

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.     

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.     

Molecular analysis of plasmid DNA repair within ultraviolet-irradiated Escherichia coli. II. UvrABC-initiated excision repair and photolyase-catalyzed dimer monomerization

In this study, a novel approach to the analysis of DNA repair in Escherichia coli was employed which allowed the first direct determination of the mechanisms by which endogenous DNA repair enzymes encounter target sites in vivo. An in vivo plasmid DNA repair analysis was employed to discriminate between two possible mechanisms of target site location: a processive DNA scanning mechanism or a distributive random diffusion mechanism. The results demonstrate that photolyase acts by a distributive mechanism within E. coli. In contrast, UvrABC-initiated excision repair occurs by a limited processive DNA scanning mechanism. A majority of the dimer sites on a given plasmid molecule were repaired prior to the dissociation of the UvrABC complex. Furthermore, plasmid DNA repair catalyzed by the UvrABC complex occurs without a detectable accumulation of nicked plasmid intermediates despite the fact that the UvrABC complex generates dual incisions in the DNA at the site of a pyrimidine dimer. Therefore, the binding or assembly of the UvrABC complex on DNA at the site of a pyrimidine dimer represents the rate-limiting step in the overall process of UvrABC-initiated excision repair in vivo.     

Catalytic sites for 3' and 5' incision of Escherichia coli nucleotide excision repair are both located in UvrC

Nucleotide excision repair in Escherichia coli is a multistep process in which DNA damage is removed by incision of the DNA on both sides of the damage, followed by removal of the oligonucleotide containing the lesion. The two incision reactions take place in a complex of damaged DNA with UvrB and UvrC. It has been shown (Lin, J. -J., and Sancar, A. (1992) J. Biol. Chem. 267, 17688-17692) that the catalytic site for incision on the 5' side of the damage is located in the UvrC protein. Here we show that the catalytic site for incision on the 3' side is in this protein as well, because substitution R42A abolishes 3' incision, whereas formation of the UvrBC-DNA complex and the 5' incision reaction are unaffected. Arg(42) is part of a region that is homologous to the catalytic domain of the homing endonuclease I-TevI. We propose that the UvrC protein consists of two functional parts, with the N-terminal half for the 3' incision reaction and the C-terminal half containing all the determinants for the 5' incision reaction.     

Interaction of the UvrABC endonuclease with DNA containing a psoralen monoadduct or cross-link. Differential effects of superhelical density and comparison of preincision complexes.

The effect of negative supercoiling on UvrABC incision of covalently closed duplex DNA circles containing either a furan-side monoadduct or a cross-link of 4'-hydroxymethyl-4,5',8-trimethylpsoralen at a unique site was examined. The rate of UvrABC incision of these DNA substrates was measured as a function of superhelical density, sigma, for values of sigma between 0 and -0.050. The monoadducted DNA substrate was incised at close to the maximum rate at all superhelical densities, with only a slight stimulation of activity between sigma = 0 and -0.035. In contrast, efficient UvrABC incision of the cross-linked DNA substrate required the DNA to be underwound, and activity showed a linear dependence on superhelical density up to sigma = -0.035. DNase I protection studies show that in the presence of both UvrA and UvrB a protein complex binds to the site of a psoralen monoadduct or cross-link in linear DNA. This UvrA-UvrB-dependent complex binds with similar affinity to both the monoadducted and the cross-linked DNA helices. However, differences in the DNase I footprint on these two DNA substrates indicate that the interaction of this protein complex is different at these two lesions. The addition of UvrC to linear DNA molecules that are saturated at the site of the lesion with the UvrA-UvrB-dependent complex resulted in efficient nicking of the monoadducted DNA, but not the cross-linked DNA. Thus, the properties of a DNA lesion site that lead to UvrAB recognition and binding are not necessarily sufficient to allow incision when all three Uvr subunits are present. We propose that after recognition and binding of a lesion site by the UvrAB complex and prior to incision, the damaged DNA helix undergoes a conformational change such as unwinding or melting that is induced by the lesion-bound Uvr complex.     

Two forms of UvrC protein with different double-stranded DNA binding affinities

Using phosphocellulose followed by single-stranded DNA-cellulose chromatography for purification of UvrC proteins from overproducing cells, we found that UvrC elutes at two peaks: 0.4 m KCl (UvrCI) and 0.6 m KCl (UvrCII). Both forms of UvrC have a major peptide band (>95%) of the same molecular weight and identical N-terminal amino acid sequences, which are consistent with the initiation codon being at the unusual GTG site. Both forms of UvrC are active in incising UV-irradiated, supercoiled phiX-174 replicative form I DNA in the presence of UvrA and UvrB proteins; however, the specific activity of UvrCII is one-fourth that of UvrCI. The molecular weight of UvrCII is four times that of UvrCI on the basis of results of size exclusion chromatography and glutaraldehyde cross-linking reactions, indicating that UvrCII is a tetramer of UvrCI. Functionally, these two forms of UvrC proteins can be distinguished under reaction conditions in which the protein/nucleotide molar ratio is >0.06 by using UV-irradiated, (32)P-labeled DNA fragments as substrates; under these conditions UvrCII is inactive in incision, but UvrCI remains active. The activity of UvrCII in incising UV-irradiated, (32)P- labeled DNA fragments can be restored by adding unirradiated competitive DNA, and the increased level of incision corresponds to a decreased level of UvrCII binding to the substrate DNA. The sites of incision at the 5' and 3' sides of a UV-induced pyrimidine dimer are the same for UvrCI and UvrCII. Nitrocellulose filter binding and gel retardation assays show that UvrCII binds to both UV-irradiated and unirradiated double-stranded DNA with the same affinity (K(a), 9 x 10(8)/m) and in a concentration-dependent manner, whereas UvrCI does not. These two forms of UvrC were also produced by the endogenous uvrC operon. We propose that UvrCII-DNA binding may interfere with Uvr(A)(2)B-DNA damage complex formation. However, because of its low copy number and low binding affinity to DNA, UvrCII may not interfere with Uvr(A)(2)B-DNA damage complex formation in vivo, but instead through double-stranded DNA binding UvrCII may become concentrated at genomic areas and therefore may facilitate nucleotide excision repair.     

Structural insights into the first incision reaction during nucleotide excision repair

Nucleotide excision repair is a highly conserved DNA repair mechanism present in all kingdoms of life. The incision reaction is a critical step for damage removal and is accomplished by the UvrC protein in eubacteria. No structural information is so far available for the 3' incision reaction. Here we report the crystal structure of the N-terminal catalytic domain of UvrC at 1.5 A resolution, which catalyzes the 3' incision reaction and shares homology with the catalytic domain of the GIY-YIG family of intron-encoded homing endonucleases. The structure reveals a patch of highly conserved residues surrounding a catalytic magnesium-water cluster, suggesting that the metal binding site is an essential feature of UvrC and all GIY-YIG endonuclease domains. Structural and biochemical data strongly suggest that the N-terminal endonuclease domain of UvrC utilizes a novel one-metal mechanism to cleave the phosphodiester bond.     

The isomerization of the UvrB-DNA preincision complex couples the UvrB and UvrC activities

In Escherichia coli nucleotide excision repair, the UvrB-DNA preincision complex plays a key role, linking adduct recognition to incision. We previously showed that the efficiency of the incision is inversely related to the stability of the preincision complex. We postulated that an isomerization reaction converts [UvrB-DNA], stable but incompetent for incision, into the [UvrB-DNA]' complex, unstable and competent for incision. Here, we identify two parameters, negative supercoiling and presence of a nick at the fifth phosphodiester bond 3' to the lesion, that accelerate the isomerization leading to an increasing incision efficiency. We also show that the [UvrB-DNA] complex is more resistant to a salt concentration increase than the [UvrB-DNA]' complex. Finally, we report that the [UvrB-DNA]' is recognized by UvrC. These data suggest that the isomerization reaction leads to an exposure of single-stranded DNA around the lesion. This newly exposed single-stranded DNA serves as a binding site and substrate for the UvrC endonuclease. We propose that the isomerization reaction is responsible for coupling UvrB and UvrC activities and that this reaction corresponds to the binding of ATP.     

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.     

The C-terminal region of the Escherichia coli UvrC protein, which is homologous to the C-terminal region of the human ERCC1 protein, is involved in DNA binding and 5'-incision.

The incisions in the DNA at the 3'- and 5'-side of a DNA damage during nucleotide excision repair in Escherichia coli occur in a complex consisting of damaged DNA, UvrB and UvrC. The exact requirements for the two incision events, however, are different. It has previously been shown that the 3'-incision requires the interaction between the C-terminal domain of UvrB and a homologous region in UvrC. This interaction, however, is dispensable for the 5'-incision. Here we show that the C-terminal domain of the UvrC protein is essential for the 5'-incision, whereas this domain can be deleted without affecting the 3'-incision. The C-terminal domain of UvrC is homologous with the C-terminal part of the ERCC1 protein which, in a complex with XPF, is responsible for the 5'-incision reaction in human nucleotide excision repair. Both in the UvrC and the ERCC1 domain a Helix-hairpin-Helix (HhH) motif can be indicated, albeit at different positions. Such a motif also has been found in a large variety of DNA binding proteins and it has been suggested to form a structure involved in non-sequence-specific DNA binding. In contrast to the full length UvrC protein, a truncated UvrC protein (UvrC554) lacking the entire ERCC1 homology including the HhH motif no longer binds to ssDNA. Analysis of protein-DNA complexes using bandshift experiments showed that this putative DNA binding domain of UvrC is required for stabilisation of the UvrBC-DNA complex after the 3'-incision has taken place. We propose that after the initial 3'-incision the HhH motif recognises a specific DNA structure, thereby positioning the catalytic site for the subsequent 5'-incision reaction.     

Consequences of molecular engineering enhanced DNA binding in a DNA repair enzyme.

Facilitated one-dimensional diffusion is a general mechanism utilized by several DNA-interactive proteins as they search for their target sites within large domains of nontarget DNA. T4 endonuclease V is a protein which scans DNA in a nonspecifically bound state and processively incises DNA at ultraviolet (UV)-induced pyrimidine dimer sites. An electrostatic contribution to this mechanism of target location has been established. Previous studies indicate that a decrease in the affinity of endonuclease V for nontarget DNA results in a decreased ability to scan DNA and a concomitant decrease in the ability to enhance UV survival in repair-deficient Escherichia coli. This study was designed to question the contrasting effect of an increase in the affinity of endonuclease V for nontarget DNA. With this as a goal, a gradient of increasingly basic amino acid content was created along a proposed endonuclease V-nontarget DNA interface. This incremental increase in positive charge correlated with the stepwise enhancement of nontarget DNA binding, yet inversely correlated with enhanced UV survival in repair-deficient E. coli. Further analysis suggests that the observed reduction in UV survival is consistent with the hypothesis that enhanced nontarget DNA affinity results in reduced pyrimidine dimer-specific recognition and/or binding. The net effect is a reduction in the efficiency of pyrimidine dimer incision.     

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.     

Structure of the C-terminal half of UvrC reveals an RNase H endonuclease domain with an Argonaute-like catalytic triad

Removal and repair of DNA damage by the nucleotide excision repair pathway requires two sequential incision reactions, which are achieved by the endonuclease UvrC in eubacteria. Here, we describe the crystal structure of the C-terminal half of UvrC, which contains the catalytic domain responsible for 5' incision and a helix-hairpin-helix-domain that is implicated in DNA binding. Surprisingly, the 5' catalytic domain shares structural homology with RNase H despite the lack of sequence homology and contains an uncommon DDH triad. The structure also reveals two highly conserved patches on the surface of the protein, which are not related to the active site. Mutations of residues in one of these patches led to the inability of the enzyme to bind DNA and severely compromised both incision reactions. Based on our results, we suggest a model of how UvrC forms a productive protein-DNA complex to excise the damage from DNA.     

Modulation of the DNA scanning activity of the Micrococcus luteus UV endonuclease

Micrococcus luteus UV endonuclease incises DNA at the sites of ultraviolet (UV) light-induced pyrimidine dimers. The mechanism of incision has been previously shown to be a glycosylic bond cleavage at the 5'-pyrimidine of the dimer followed by an apyrimidine endonuclease activity which cleaves the phosphodiester backbone between the pyrimidines. The process by which M. luteus UV endonuclease locates pyrimidine dimers within a population of UV-irradiated plasmids was shown to occur, in vitro, by a processive or "sliding" mechanism on non-target DNA as opposed to a distributive or "random hit" mechanism. Form I plasmid DNA containing 25 dimers per molecule was incubated with M. luteus UV endonuclease in time course reactions. The three topological forms of plasmid DNA generated were analyzed by agarose gel electrophoresis. When the enzyme encounters a pyrimidine dimer, it is significantly more likely to make only the glycosylase cleavage as opposed to making both the glycosylic and phosphodiester bond cleavages. Thus, plasmids are accumulated with many alkaline-labile sites relative to single-stranded breaks. In addition, reactions were performed at both pH 8.0 and pH 6.0, in the absence of NaCl, as well as 25,100, and 250 mM NaCl. The efficiency of the DNA scanning reaction was shown to be dependent on both the ionic strength and pH of the reaction. At low ionic strengths, the reaction was shown to proceed by a processive mechanism and shifted to a distributive mechanism as the ionic strength of the reaction increased. Processivity at pH 8.0 is shown to be more sensitive to increases in ionic strength than reactions performed at pH 6.0.