den V gene of bacteriophage T4 determines a DNA glycosylase specific for pyrimidine dimers in DNA.

Endonuclease V of bacteriophage T4 has been described as an enzyme, coded for by the denV gene, that incises UV-irradiated DNA. It has recently been proposed that incision of irradiated DNA by this enzyme and the analogous "correndonucleases" I and II of Micrococcus luteus requires the sequential action of a pyrimidine dimer-specific DNA glycosylase and an apyrimidinic/apurinic endonuclease. In support of this two-step mechanism, we found that our preparations of T4 endonuclease V contained a DNA glycosylase activity that produced alkali-labile sites in irradiated DNA and an apyrimidinic/apurinic endonuclease activity that converted these sites to nicks. Both activities could be detected in the presence of 10 mM EDTA. In experiments designed to determine which of the activities is coded by the denV gene, we found that the glycosylase was more heat labile in extracts of Escherichia coli infected with either of two thermosensitive denV mutants than in extracts of cells infected with wild-type T4. In contrast, apyrimidinic/apurinic endonuclease activity was no more heat labile in extracts of the former than in extracts of the latter. Our results indicate that the denV gene codes for a DNA glycosylase specific for pyrimidine dimers.     

Binding of T4 endonuclease V to deoxyribonucleic acid irradiated with ultraviolet light

Endonuclease V of bacteriophage T4 binds to UV-irradiated deoxyribonucleic acid (DNA) but not to unirradiated DNA. We have developed an assay to detect this binding, based on the retention of enzyme--DNA complexes on nitrocellulose filters. The amount of complex retained, ascertained by using radioactive DNA, is a measure of T4 endonuclease V activity. The assay is simple, rapid, and specific, which makes it useful for detecting T4 endonuclease V activity both in crude lysates and in purified preparations. We have used it to monitor enzyme activity during purification and to study binding of the enzyme to DNA under conditions that minimize the ability of the enzyme to nick DNA. From our data we conclude that (1) T4 endonuclease V binds to UV-irradiated DNA but not to DNA that has been previously incised by the endonuclease, (2) equilibrium between the free and complexed form of the enzyme is attained under our reaction conditions, (3) dissociation of enzyme--DNA complexes is retarded by sodium cyanide, and (4) retention of enzyme--DNA complexes on nitrocellulose filters is enhanced by high concentrations of saline--citrate.     

Hydration at the TD Damaged Site of DNA and its Role in the Formation of Complex with T4 Endonuclease V

Abstract

An analysis of the distribution of water around DNA surface focusing on the role of the distribution of water molecules in the proper recognition of damaged site by repair enzyme T4 Endonuclease V was performed. The native DNA dodecamer, dodecamer with the thymine dimer (TD) and complex of DNA and part of repair enzyme T4 Endonuclease V were examined throughout the 500 ps of molecular dynamics simulation. During simulation the number of water molecules close to the DNA atoms and the residence time were calculated. There is an increase in number of water molecules lying in the close vicinity to TD if compared with those lying close to two native thymines (TT). Densely populated area with water molecules around TD is one of the factors detected by enzyme during scanning process. The residence time was found higher for molecule of the complex and the six water molecules were found occupying the stabile positions between the TD and catalytic center close to atoms P, C3′ and N3. These molecules originate water mediated hydrogen bond network that contribute to the stability of complex required for the onset of repair process.     

Action of T4 endonuclease V on DNA containing various photoproducts

The action of T4 endonuclease V on DNA containing various photoproducts was investigated. (1) The enzyme introduced strand breaks in DNA from ultraviolet-irradiated vegetative cells of Bacillus subtilis but not in DNA from irradiated spores of the same organism. DNA irradiated with long wavelength (360 nm peak) ultraviolet light in the presence of 4,5',8-trimethylpsoralen was not attacked by the enzyme. These results indicate that 5-thyminyl 5,6-dihydrothymine (spore photoproduct) and psoralen mediated cross-links in DNA are not recognized by T4 endonuclease V. (2) DNA of phage PBS1, containing uracil in place of thymine, and DNA of phage SPO1, containing hydroxymethyluracil in place of thymine, were fragmented by the enzyme when the DNA's had been irradiated with ultraviolet light. T4 endonuclease V seems to act on DNA with pyrimidine dimers whether the dimers contain thymine residues or not.     

Repair of deaminated bases in DNA

Deamination of DNA bases can occur spontaneously, generating highly mutagenic lesions such as uracil, hypoxanthine, and xanthine. When cells are under oxidative stress that is induced either by oxidizing agents or by mitochondrial dysfunction, additional deamination products such as 5-hydroxymethyluracil (5-HMU) and 5-hydroxyuracil (5-OH-Ura) are formed. The cellular level of these highly mutagenic lesions is increased substantially when cells are exposed to DNA damaging agent, such as ionizing radiation, redox reagents, nitric oxide, and others. The cellular repair of deamination products is predominantly through the base excision repair (BER) pathway, a major cellular repair pathway that is initiated by lesion specific DNA glycosylases. In BER, the lesions are removed by the combined action of a DNA glycosylase and an AP endonuclease, leaving behind a one-base gap. The gapped product is then further repaired by the sequential action of DNA polymerase and DNA ligase. DNA glycosylases that recognize uracil, 5-OH-Ura, 5-HMU (derived from 5-methylcytosine) and a T/G mismatch (derived from a 5-methylcytosine/G pair) are present in most cells. Many of these glycosylases have been cloned and well characterized. In yeast and mammalian cells, hypoxanthine is efficiently removed by methylpurine N-glycosylase, and it is thought that BER might be an important pathway for the repair of hypoxanthine. In contrast, no glycosylase that can recognize xanthine has been identified in either yeast or mammalian cells. In Escherichia coli, the major enzyme activity that initiates the repair of hypoxanthine and xanthine is endonuclease V. Endonuclease V is an endonuclease that hydrolyzes the second phosphodiester bond 3' to the lesion. It is hypothesized that the cleaved DNA is further repaired through an alternative excision repair (AER) pathway that requires the participation of either a 5' endonuclease or a 3'-5' exonuclease to remove the damaged base. The repair process is then completed by the sequential actions of DNA polymerase and DNA ligase. Endonuclease V sequence homologs are present in all kingdoms, and it is conceivable that endonuclease V might also be a major enzyme that initiates the repair of hypoxanthine and xanthine in mammalian cells.     

Purification of the T4 endonuclease V

A new purification protocol has been developed for the rapid isolation to physical homogeneity of T4 endonuclease V. The enzyme was purified from an Escherichia coli strain which harbors a plasmid containing the T4 denV structural gene downstream of the lambda rightward promoter. The purification of the enzyme was monitored by pyrimidine dimer-specific nicking activity, Western blot analysis and silver or Coomassie Blue staining of SDS-polyacrylamide gels. Milligram quantities of the enzyme have been purified by the following procedure. After sonication of cells and removal of major cell debris, total protein and nucleic acids were passed over a single-stranded DNA agarose column. Endonuclease V was eluted at 650 mM KCl with a linear salt gradient yielding enzyme of approximately 20% purity and following dialysis, was applied to a chromatofocusing column. The enzyme elutes at pH 9.4 and is greater than 90% homogeneous at this step. The final purification step is CM-Sephadex chromatography which attains greater than 98% homogeneity.     

On the recognition and cleavage mechanism of Escherichia coli endodeoxyribonuclease V, a possible DNA repair enzyme

Escherichia coli endodeoxyribonuclease V acts at many sites of damage in duplex DNA, including apurinic/apyrimidinic sites, lesions induced by ultraviolet light which are not pyrimidine dimers, adducts of 7-bromomethylbenz[a]anthracene, and, as demonstrated earlier (Gates, F. T., and Linn, S. (1977a) J. Biol. Chem. 252. 1647-1653), it degrades uracil-containing duplex DNA most efficiently. The cleavage rate increases with increasing substitution of uracil for thymine in T5 DNA, with a replacement of one-eight of thymine generating the apparent maximum cleavage rate. However, the apparent reaction limit with DNA containing 3.8% of thymine replaced by uracil corresponds to cleavage at only 6% of the dUMP residues. Evidently, the enzyme recognizes some peculiarities of abnormal DNA structure, but not simply distortions, since some lesions, including pyrimidine dimers, are not substrates. Endonuclease V generates double strand breaks in a constant ratio to single strand nicks, regardless of the substrate. It degrades DNA processively, completing the digestion of one substrate molecule before proceeding to the next. The enzyme also appears to act cooperatively. Cleavage at methylbenz[a]anthracene adducts is usually or always 5' to the lesion. Endonuclease V seems well suited to act as a DNA repair enzyme, surveying the genome for structural distortions generated by lesions for which specific repair systems might not exist.     

Studies on temperature-dependent ultraviolet light-sensitive mutants of bacteriophage T4: the structural gene for T4 endonuclease V.

Mutants of bacteriophage T4 which exhibit increased sensitivity to ultraviolet radiation specifically at high temperature were isolated after mutagenesis with hydroxylamine. At 42 °C the mutants are twice as sensitive to ultraviolet light as T4D, whereas at 30 °C they exhibit survival curves almost identical to that of the wild-type strain. Complementation tests revealed that the mutants possess temperature-sensitive mutations in the v gene.Evidence is presented to show that T4 endonuclease V produced by the mutants is more thermolabile than the enzyme of the wild-type. (1) Extracts of cells infected with the mutants were capable of excising pyrimidine dimers from ultraviolet irradiated T4 DNA at 30 °C, but no selective release of dimers was induced at 42 °C. (2) Endonuclease V produced by the mutant was inactivated more rapidly than was the enzyme from T4D-infected cells when the purified enzymes were incubated in a buffer at 42 °C. From these results it is evident that the v gene is the structural gene for T4 endonuclease V, which plays an essential role in the excision-repair of ultraviolet light-damaged DNA.The time of action of the repair endonuclease was determined by using the mutant. Survival of a temperature-sensitive v mutant, exposed to ultraviolet light, increased when infected cells were incubated at 30 °C for at least ten minutes and then transferred to 42 °C. It appears that repair of DNA proceeds during an early stage of phage development.     

Site-directed mutagenesis of the T4 endonuclease V gene: the role of arginine-3 in the target search

Endonuclease V, a pyrimidine dimer specific endonuclease in T4 bacteriophage, is able to scan DNA, recognize pyrimidine dimer photoproducts produced by exposure to ultraviolet light, and effectively incise DNA through a two-step mechanism at the damaged bases. The interaction of endonuclease V with nontarget DNA is thought to occur via electrostatic interactions between basic amino acids and the acidic phosphate DNA backbone. Arginine-3 was chosen as a potential candidate for involvement in this protein-nontarget DNA interaction and was extensively mutated to assess its role. The mutations include changes to Asp, Glu, Leu, and Lys and deleting it from the enzyme. Deletion of Arg-3 resulted in an enzyme that retained marginal levels of AP specificity, but no other detectable activity. Charge reversal to Glu-3 and Asp-3 results in proteins that exhibit AP-specific nicking and low levels of dimer-specific nicking. These enzymes are incapable of affecting cellular survival of repair-deficient Escherichia coli after irradiation. Mutations of Arg-3 to Lys-3 or Leu-3 also are unable to complement repair-deficient E. coli. However, these two proteins do exhibit a substantial level of in vitro dimer- and AP-specific nicking. The mechanism by which the Leu-3 and Lys-3 mutant enzymes locate pyrimidine dimers within a population of heavily irradiated plasmid DNA molecules appears to be significantly different from that for the wild-type enzyme. The wild-type endonuclease V processively incises all dimers on an individual plasmid prior to dissociation from that plasmid and subsequent reassociation with other plasmids, yet neither of these mutants exhibits any of the characteristics of this processive nicking activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resolution of branched DNA substrates by T7 endonuclease I and its inhibition

Endonuclease I is a multipurpose enzyme implicated in the breakdown of host DNA, packaging of phage DNA, and recombination during the lytic cycle of bacteriophage T7. We investigate here some aspects of the substrate requirements for its activity in resolving branched intermediates similar to Holliday junctions (Holliday, R. (1964) Genet. Res. 5, 282-304) that arise in recombination. The enzyme is able to resolve branched substrates containing very short duplex arms: 4 base pairs suffice. It cleaves 5' to the branch, with a distinct preference for the non-crossover strands in Holliday-like model junctions. Ligands that interact strongly with the branch site can inhibit the enzyme, with KI values in the 10-50 microM range.     

5' leads to 3'-Exonucleases of bacteriophage T4.

Two enzyme activities which release nucleotides preferentially from the 5' termini of DNA were found in T4-infected Escherichia coli. Since no corresponding activities were found in uninfected cells, the activities appeared to be induced by T4. Both activities are capable of excising pyrimidine dimers from ultraviolet-irradiated DNA which has been treated with T4 endonuclease V. One of the activities , referred to as T4 exonuclease B, was purified 400-fold from an extract of T4v 1- infected cells. The enzyme initiates hydrolysis of DNA specifically at the 5' termini to yield products which are mainly oligonucleotides of varying length. The hydrolysis reaction proceeds in a limited manner. The enzyme shows optimal activity at pH 7.0 and absolutely requires Mg2+. The molecular weight of the enzyme , as estimated by gel filtration, is approximately 35,000. Another activity, referred to as T4 exonuclease C, was purified 240-fold from the extract. This activity also excises pyrimidine dimers from ultraviolet-irradiated, incised DNA and releases nucleotides at 5' termini. It has a pH optimum at 7.5 and requires Mg2+. The molecular weight of the enzyme is approximately 20,000.     

Cyclobutane pyrimidine dimer formation is a molecular trigger for solar-simulated ultraviolet radiation-induced suppression of memory immunity in humans.

We tested the hypothesis that DNA is a target for solar-simulated ultraviolet radiation (ssUVR)-induced suppression of the reactivation of memory immunity in humans. T4N5 liposomes contain the DNA repair enzyme T4 endonuclease V. This cleaves DNA at the site of ultraviolet radiation (UVR)-induced cyclobutane pyrimidine dimers (CPD), initiating DNA repair. It has previously been used to show that CPDs are a key molecular trigger for UVR-induced immunosuppression in mice. To determine whether CPD formation is involved in UVR immunosuppression in humans, nickel-allergic volunteers were irradiated with a range of doses of ssUVR. T4N5 or empty liposomes were then applied after irradiation. Nickel-induced recall immunity was assessed by reflectance spectrometry. T4N5 liposomes inhibited immunosuppression and prevented ssUVR from reducing the number of epidermal dendritic cells. T4N5 liposomes also reduced macrophage infiltration into irradiated epidermis. These studies show that enhanced removal of CPDs from human skin protects from immunosuppression, hence demonstrating that these photolesions are an important molecular event in ssUVR-induced immunosuppression in humans. CPDs also triggered loss of dendritic cells and infiltration by macrophages. It is possible that these changes to antigen presenting cells contribute to ssUVR induced suppression of recall immunity to nickel in humans.     

Switching base preferences of mismatch cleavage in endonuclease V: an improved method for scanning point mutations

Endonuclease V (endo V) recognizes a broad range of aberrations in DNA such as deaminated bases or mismatches. It nicks DNA at the second phosphodiester bond 3′ to a deaminated base or a mismatch. Endonuclease V obtained from Thermotoga maritima preferentially cleaves purine mismatches in certain sequence context. Endonuclease V has been combined with a high-fidelity DNA ligase to develop an enzymatic method for mutation scanning. A biochemical screening of site-directed mutants identified mutants in motifs III and IV that altered the base preferences in mismatch cleavage. Most profoundly, a single alanine substitution at Y80 position switched the enzyme to essentially a C-specific mismatch endonuclease, which recognized and cleaved A/C, C/A, T/C, C/T and even the previously refractory C/C mismatches. Y80A can also detect the G13D mutation in K-ras oncogene, an A/C mismatch embedded in a G/C rich sequence context that was previously inaccessible using the wild-type endo V. This investigation offers insights on base recognition and active site organization. Protein engineering in endo V may translate into better tools in mutation recognition and cancer mutation scanning.     

Purification and some properties of deoxyribonuclease whose synthesis is controlled by gene 49 of bacteriophage T4.

An enzyme which specifically cleaves very-fast-sedimenting DNA of bacteriophage T4 is synthesized after infection of T4, and its synthesis is controlled by gene 49 [1,2]. This enzyme has been proved to be a DNase [2]. We have purified this DNase 3000-fold from extracts of E. coli infected with T4. The purified preparation was practically free from other DNases, and the DNase activity was not detectable in cells infected with a mutant defective in gene 49. The enzyme activity from cells infected with a temperature-sensitive mutant of gene 49 was also temperature-sensitive, suggesting strongly that gene 49 is a structural gene of the DNase. The molecular weight of the wild-type enzyme was estimated to be 50 x 10(3) by gel filtration chromatography. The purified DNase did not cleave native and denatured DNAs of T3 and T4, but cleaved renatured T3 DNA with enzymatically fragmented T3 DNA, indicating that gaps in the DNA duplex are structures susceptible to the DNase. Cleavage of the hybridized T3 DNA occurred when the fragmented DNA was phosphorylated at either the 3' or 5'-strand termini.     

Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis

Endonuclease IV encoded by denB of bacteriophage T4 is implicated in restriction of deoxycytidine (dC)-containing DNA in the host Escherichia coli. The enzyme was synthesized with the use of a wheat germ cell-free protein synthesis system, given a lethal effect of its expression in E.coli cells, and was purified to homogeneity. The purified enzyme showed high activity with single-stranded (ss) DNA and denatured dC-substituted T4 genomic double-stranded (ds) DNA but exhibited no activity with dsDNA, ssRNA or denatured T4 genomic dsDNA containing glucosylated deoxyhydroxymethylcytidine. Characterization of Endo IV activity revealed that the enzyme catalyzed specific endonucleolytic cleavage of the 5′ phosphodiester bond of dC in ssDNA with an efficiency markedly dependent on the surrounding nucleotide sequence. The enzyme preferentially targeted 5′-dTdCdA-3′ but tolerated various combinations of individual nucleotides flanking this trinucleotide sequence. These results suggest that Endo IV preferentially recognizes short nucleotide sequences containing 5′-dTdCdA-3′, which likely accounts for the limited digestion of ssDNA by the enzyme and may be responsible in part for the indispensability of a deficiency in denB for stable synthesis of dC-substituted T4 genomic DNA.     

Replacing tryptophan-128 of T4 endonuclease V with a serine residue results in decreased enzymatic activity in vitro and in vivo.

Endonuclease V of bacteriophage T4 possesses two enzymatic activities, a DNA N-glycosylase specific for cyclobutane pyrimidine dimers (CBPD) and an associated apurinic/apyrimidinic (AP) lyase. Extensive structural and functional studies of endonuclease V have revealed that specific amino acids are associated with these two activities. Controversy still exists regarding the role of the aromatic amino acid stretch close to the carboxyl terminus, in particular the tryptophan at position 128. We have expressed wild-type and mutant W128S endonuclease V in Escherichia coli from an inducible tac promoter. Purified W128S endonuclease V demonstrated substantially decreased N-glycosylase (approximately 5-fold) and AP lyase (10- to 20-fold) activities in vitro compared to the wild-type enzyme when a UV-irradiated poly(dA)-poly(dT) substrate was used. However, a much smaller difference in AP lyase activity between the two forms was observed with a site-specific abasic oligonucleotide. The difference in enzymatic activity was qualitatively, but not quantitatively, reflected in the survival of UV-irradiated bacteria, that is the W128S cells were slightly less UV resistant than wild-type cells. No difference was observed in the complementation of UV repair using UV-damaged denV- T4 phage. A more pronounced difference between the wild-type and W128S proteins was observed in human xeroderma pigmentosum group A cells by host-cell reactivation of a UV-irradiated reporter gene. The relatively large discrepancy between the in vitro and in vivo results observed with bacteria may be because saturated levels of DNA repair are obtained in vivo with relatively low levels of endonuclease V. However, under limiting in vitro conditions and in human cells in vivo a considerable difference between the W128S mutant and wild-type endonuclease V activities can be detected. Our results demonstrate that tryptophan-128 is important for endonuclease V activity.     

T7-induced DNA polymerase. Characterization of associated exonuclease activities and resolution into biologically active subunits.

Bacteriophage T7-induced DNA polymerase has been isolated by a procedure suitable for large scale use and which yields near homogeneous enzyme. In addition to previously described DNA polymerase activity and 3' to 5' exonucleolytic activity on single stranded DNA (Grippo, P., and Richardson, C. C. (1971) J. Biol. Chem. 246, 6867-6873), the enzyme also possesses a highly active exonuclease which hydrolyzes duplex substrates with 3' to 5' directionality. The native polymerase has been dissociated using 6 M guanidine HCl and resolved into biologically active subunits: T7 gene 5 protein and Escherichia coli thioredoxin. The phage-specified subunit obtained by this procedure is deficient in DNA polymerase and double strand exonuclease activities, with deficiencies in these activities being apparent at the level of a single turnover. However, it possesses near normal levels of a single strand hydrolytic activity which is identical to that associated with the native polymerase with respect to substrate specificity and suppression of hydrolysis by low levels of deoxyribonucleoside 5'-triphosphates. Thioredoxin forms a molecular complex with the T7 gene 5 protein, and addition of the host protein restores restores DNA polymerase and double strand exonuclease activities to near normal levels.     

T4 endonuclease V exists in solution as a monomer and binds to target sites as a monomer

Endonuclease V, a N-glycosylase/lyase from T4 bacteriophage that initiates the repair of cyclobutane pyrimidine dimers in DNA, has been reported to form a monomer-dimer equilibrium in solution [Nickell and Lloyd (1991) Biochemistry 30, 8638], although the enzyme has only been crystallized in the absence of substrate as a monomer [Morikawa et al. (1992) Science 256, 523]. In this study, analytical gel filtration and sedimentation equilibrium techniques were used to rigorously characterize the association state of the enzyme in solution. In contrast to the previous report, at 100 mM KCl endonuclease V was found to exist predominantly as a monomer in solution by both of these techniques; no evidence for dimerization was seen. To characterize the oligomeric state of the enzyme at its target sites on DNA, the enzyme was bound to oligonucleotides containing a single site specific pyrimidine dimer or tetrahydrofuran residue. These complexes were analyzed by nondenaturing gel electrophoresis at various acrylamide concentrations in order to determine the molecular weights of the enzyme-DNA complexes. The results from these experiments demonstrate that endonuclease V binds to cyclobutane pyrimidine dimer and tetrahydrofuran site containing DNA as a monomer.     

Selective metal binding to Cys-78 within endonuclease V causes an inhibition of catalytic activities without altering nontarget and target DNA binding

T4 endonuclease V is a pyrimidine dimer-specific DNA repair enzyme which has been previously shown not to require metal ions for either of its two catalytic activities or its DNA binding function by virtue of its ability to function in the presence of metal-chelating agents. However, we have investigated whether the single cysteine within the enzyme was able to bind metal salts and influence the various activities of this repair enzyme. A series of metals (Hg2+, Ag+, Cu+) were shown to inactivate both endonuclease Vs pyrimidine dimer-specific DNA glycosylase activity and the subsequent apurinic nicking activity. The binding of metal to endonuclease V did not interfere with nontarget DNA scanning or pyrimidine dimer-specific binding. The Cys-78 codon within the endonuclease V gene was changed by oligonucleotide site-directed mutagenesis to Thr-78 and Ser-78 in order to determine whether the native cysteine was directly involved in the enzyme's DNA catalytic activities and whether the cysteine was primarily responsible for the metal binding. The mutant enzymes were able to confer enhanced ultraviolet light (UV) resistance to DNA repair-deficient Escherichia coli at levels equal to that conferred by the wild type enzyme. The C78T mutant enzyme was purified to homogeneity and shown to be catalytically active on pyrimidine dimer-containing DNA. The catalytic activities of the C78T mutant enzyme were demonstrated to be unaffected by the addition of Hg2+ or Ag+ at concentrations 1000-fold greater than that required to inhibit the wild type enzyme. These data suggest that the cysteine is not required for enzyme activity but that the binding of certain metals to that amino acid block DNA incision by either preventing a conformational change in the enzyme after it has bound to a pyrimidine dimer or sterically interfering with the active site residue's accessibility to the pyrimidine dimer.     

Detecting ultraviolet damage in single DNA molecules by atomic force microscopy

We report detection and quantification of ultraviolet (UV) damage in DNA at a single molecule level by atomic force microscopy (AFM). By combining the supercoiled plasmid relaxation assay with AFM imaging, we find that high doses of medium wave ultraviolet (UVB) and short wave ultraviolet (UVC) light not only produce cyclobutane pyrimidine dimers (CPDs) as reported but also cause significant DNA degradation. Specifically, 12.5 kJ/m(2) of UVC and 165 kJ/m(2) of UVB directly relax 95% and 78% of pUC18 supercoiled plasmids, respectively. We also use a novel combination of the supercoiled plasmid assay with T4 Endonuclease V treatment of irradiated plasmids and AFM imaging of their relaxation to detect damage caused by low UVB doses, which on average produced approximately 0.5 CPD per single plasmid. We find that at very low UVB doses, the relationship between the number of CPDs and UVB dose is almost linear, with 4.4 CPDs produced per Mbp per J/m(2) of UVB radiation. We verified these AFM results by agarose gel electrophoresis separation of UV-irradiated and T4 Endonuclease V treated plasmids. Our AFM and gel electrophoresis results are consistent with the previous result obtained using other traditional DNA damage detection methods. We also show that damage detection assay sensitivity increases with plasmid size. In addition, we used photolyase to mark the sites of UV lesions in supercoiled plasmids for detection and quantification by AFM, and these results were found to be consistent with the results obtained by the plasmid relaxation assay. Our results suggest that AFM can supplement traditional methods for high resolution measurements of UV damage to DNA.