Ultraviolet-induced thymine hydrates in DNA are excised by bacterial and human DNA glycosylase activities

Ultraviolet irradiation of DNA results in various pyrimidine modifications. We studied the excision of an ultraviolet thymine photoproduct by Escherichia coli endonuclease III and by a preparation of human WI-38 cells. These enzymes cleave UV-irradiated DNA at apyrimidinic sites formed by glycosylic removal of the photoproduct. Poly(dA-[3H]dT).poly(dA-[3H]dT) was UV irradiated and incubated with purified E. coli endonuclease III. 3H-Containing material was released in a manner consistent with Michaelis-Menten kinetics. This 3H-labeled material was determined to be a mixture of thymine hydrates (6-hydroxy-5,6-dihydrothymine), separable from unmodified thymine by chromatography in three independent systems. Both cis-thymine hydrate and trans-thymine hydrate were chemically and photochemically synthesized. These coeluted with the enzyme-released 3H-containing material. No thymine glycol was released from the UV-irradiated polymer. Similar results were obtained with extracts of WI-38 cells as the enzyme source. The release of thymine hydrates by both glycosylase activities was directly proportional to the amount of enzyme and the irradiation dose to the DNA substrate. These results demonstrate the modified thymine residues recognized and excised by endonuclease III and the human enzyme to be a mixture of cis-thymine hydrate and trans-thymine hydrate. The reparability of these thymine hydrates suggests that they are stable in DNA and therefore potentially genotoxic.     

Gamma endonuclease of Micrococcus luteus: action on irradiated DNA

Gamma endonuclease is a Mg2+-independent enzyme of Micrococcus luteus that recognizes and cleaves DNA at a variety of altered pyrimidines produced by ionizing radiation. The production of enzyme-recognizable sites (ERS) by ionizing radiation under different irradiation conditions was measured. Ionizing radiation produced the greatest number of ERS when irradiations were performed under anoxic conditions in the presence of the free radical scavenger KI. Since dihydrothymine is a major pyrimidine lesion produced in DNA during anoxic irradiation, the ability of gamma endonuclease to excise this lesion was assessed. Dihydrothymine was released from DNA irradiated under anoxic conditions in a radiation dose-dependent manner, consistent with gamma endonuclease's known DNA glycosylase activity. Gamma endonuclease was also shown to cleave heavily uv-irradiated DNA. When the sequence specificity of gamma-endonuclease cleavage was studied using uv-irradiated DNA, cleavage was seen specifically at cytosines. The identity of this enzyme-recognizable cytosine photoproduct is not known.     

Purification and characterization of an endonuclease from calf thymus acting on irradiated DNA.

An endonuclease acting on DNA exposed to ultraviolet light or gamma-rays has been extensively purified from calf thymus. The enzyme has a pH optimum at pH 7.0-7.5, acts with equal efficiency in the presence of EDTA or divalent cations (Mg-2+ or Ca-2+), is inhibited by NaCl and tRNA and is inactivated by incubation at 50 degrees C. Its molecular weight, determined by Sephadex chromatography or sodium dodecylsulfate gel electrophoresis, is approx. 30 000. The enzyme catalyzes the formation of breaks with 5'-phosphate termini in double-stranded DNA irradiated with ultraviolet or gamma-rays. It does not act on unirradiated DNA or denatured DNA. Since in all these properties the enzymatic activity on ultraviolet- and gamma-irradiated DNA behaved similarly and since the two activities cochromatographed in all systems used during purification, we conclude that they are associated with the same protein. The site of action of the enzyme in ultraviolet-irradiated DNA is a photoproduct other than pyrimidine dimers. Such a photoproduct can also be induced by irradiation of the DNA in vivo, i.e. within the cells.     

Excision of cytosine hydrates from Z-DNA.

Ultraviolet irradiation of DNA produces cytosine hydrate, released as a free base by E. coli endonuclease III. Cytosine hydrate excision was investigated by assaying photoproduct release from cytosine-radiolabeled, irradiated poly(dG-dC):poly(dG-dC). Conformational shifts between B-DNA and Z-DNA were affected by heating the polymer in either nickel chloride or cobaltous chloride, and were determined by circular dichroism. Rates of enzymic cytosine hydrate release did not differ between the different substrate conformations. Irradiation of left-handed poly(dG-dC):poly(dG-dC) resulted in cytosine hydrate formation. Therefore, neither formation nor enzymic excision of ultraviolet-induced cytosine hydrates are substantially affected by these DNA conformational states.     

Raman spectroscopic analysis of the effect of ultraviolet irradiation on calf thymus DNA

Raman spectroscopy was used for the first time to detect the effect of independent UVA (ultraviolet-A: 320-400nm) and UVB (ultraviolet-B: 280-320 nm) irradiation on the calf thymus DNA in aqueous solution. After both UVA and UVB irradiation for 1h or 3h, the damage to the conformation of DNA was moderate, but the reduction of the B-form DNA component was obvious. Both UVA and UVB caused significant damage to the deoxyribose moiety and bases, among which the pyrimidine base pairs were more seriously affected. There appeared to be preferential damaging sites on DNA molecules caused by UVA and UVB irradiation. UVA irradiation caused more damage to the deoxyribose than UVB irradiation, while UVB irradiation caused more significant damage to the pyrimidine moiety than UVA irradiation. After UVB irradiation for 3h, unstacking of the AT base pairs and the cytosine ring took place, severe damage to the thymine moiety occurred, and some base pairs were modified. Moreover, with either UVA or UVB irradiation for 3h,the photoreactivation of DNA occurred. The damage to the DNA caused by UVB was immediate, while the damage caused by UVA was proportional to the irradiation duration. The experimental results partly indicate the formation of some cyclobutane pyrimidine dimers and (6-4) photoproducts.     

Formation and stability of repairable pyrimidine photohydrates in DNA

Ultraviolet irradiation of poly(dG-dC) and poly(dA-dU) in solution produces pyrimidine hydrates that are repaired by bacterial and mammalian DNA glycosylases [Boorstein et al. (1989) Biochemistry 28, 6164-6170]. Escherichia coli endonuclease III was used to quantitate the formation and stability of these hydrates in the double-stranded alternating copolymers poly(dG-dC) and poly(dA-dU). When poly(dG-dC) was irradiated with 100 kJ/m2 of 254-nm light at pH 8.0, 2.2% of the cytosine residues were converted to cytosine hydrate (6-hydroxy-5,6-dihydrocytosine) while 0.09% were converted to uracil hydrate (6-hydroxy-5,6-dihydrouracil). To measure the stability of these products, poly(dG-dC) was incubated in solution for up to 24 h after UV irradiation. Cytosine hydrate was stable at 4 degrees C and decayed at 25, 37, and 55 degrees C with half-lives of 75, 25, and 6 h. Uracil hydrate produced in irradiated poly(dA-dU) was stable at 4 degrees C and at 25 degrees C and decayed with a half-life of 6 h at 37 degrees C and less than 0.5 h at 55 degrees C. Uracil hydrate and uracil were also formed in irradiated poly(dG-dC). These experiments demonstrate that UV-induced cytosine hydrate may persist in DNA for prolonged time periods and also undergo deamination to uracil hydrate, which in turn undergoes dehydration to yield uracil. The formation and stability of these photoproducts in DNA may have promoted the evolutionary development of the repair enzyme endonuclease III and analogous DNA glycosylase/endonuclease activities of higher organisms, as well as the development of uracil-DNA glycosylase.     

UV-induced pyrimidine hydrates in DNA are repaired by bacterial and mammalian DNA glycosylase activities

Escherichia coli endonuclease III and mammalian repair enzymes cleave UV-irradiated DNA at AP sites formed by the removal of cytosine photoproducts by the DNA glycosylase activity of these enzymes. Poly(dG-[3H]dC) was UV irradiated and incubated with purified endonuclease III. 3H-Containing material was released in a fashion consistent with Michaelis-Menten kinetics. This 3H material was determined to be cytosine by chromatography in two independent systems and microderivatization. 3H-Containing material was not released from nonirradiated copolymer. When poly(dA-[3H]dU) was UV irradiated, endonuclease III released 3H-containing material that coeluted with uracil hydrate (6-hydroxy-5,6-dihydrouracil). Similar results are obtained by using extracts of HeLa cells. There results indicate that the modified cytosine residue recognized by endonuclease III and the mammalian enzyme is cytosine hydrate (6-hydroxy-5,6-dihydrocytosine). Once released from DNA through DNA-glycosylase action, the compound eliminates water, reverting to cytosine. This is consistent with the known instability of cytosine hydrate. The repairability of cytosine hydrate in DNA suggests that it is stable in DNA and potentially genotoxic.     

Mechanism of action of a mammalian DNA repair endonuclease

The mechanism of action of a DNA repair endonuclease isolated from calf thymus was determined. The calf thymus endonuclease possesses a substrate specificity nearly identical with that of Escherichia coli endonuclease III following DNA damage by high doses of UV light, osmium tetroxide, and other oxidizing agents. The calf thymus enzyme incises damaged DNA at sites of pyrimidines. A cytosine photoproduct was found to be the primary monobasic UV adduct. The calf thymus endonuclease and E. coli endonuclease III were found to possess similar, but not identical, DNA incision mechanisms. The mechanism of action of the calf thymus endonuclease was deduced by analysis of the 3' and 5' termini of the enzyme-generated DNA scission products with DNA sequencing methodologies and HPLC analysis of the material released by the enzyme following DNA damage. The calf thymus endonuclease removes UV light and osmium tetroxide damaged bases via an N-glycosylase activity followed by a 3' apurinic/apyrimidinic (AP) endonuclease activity. The calf thymus endonuclease also possesses a novel 5' AP endonuclease activity not possessed by endonuclease III. The product of this three-step mechanism is a nucleoside-free site flanked by 3'-and 5'-terminal phosphate groups. These results indicate the conservation of both substrate specificity and mechanism of action in the enzymatic removal of oxidative base damage between prokaryotes and eukaryotes. We propose the name redoxy endonucleases for this group of enzymes.     

The DNA damage spectrum produced by simulated sunlight

The mutagenic effects of ultraviolet and solar irradiation are thought to be due to the formation of DNA photoproducts, most notably cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)PPs). Experimental systems for determining the levels and sequence dependence of photoproduct formation in DNA have often used high doses of short-wave (UVC) irradiation. We have re-assessed this issue by using DNA sequencing technologies and different doses of UVC as well as more physiologically relevant doses of solar irradiation emitted from a solar UV simulator. It has been questioned whether hot alkali treatment can detect (6-4)PPs at all sequence positions. With high UVC doses, the sequence distribution of (6-4)PPs was virtually identical when hot alkali or UV damage endonuclease (UVDE) were used for detection, which appears to validate both methods. The (6-4)PPs form at 5'-TpC and 5'CpC sequences but very low levels are seen at all other dipyrimidines including 5'-TpT. Contrary to expectation, we find that (6-4) photoproducts form at almost undetectable levels under conditions of irradiation for up to five hours with the solar UV simulator. The same treatment produces high levels of CPDs. In addition, DNA glycosylases, which recognize oxidized and ring-opened bases, did not produce significant cleavage of sunlight-irradiated DNA. From these data, we conclude that cyclobutane pyrimidine dimers are at least 20 to 40 times more frequent than any other DNA photoproduct when DNA or cells are irradiated with simulated sunlight.     

Photoalkylated DNA and ultraviolet-irradiated DNA are incised at cytosines by endonuclease III.

Photoalkylation, the ultraviolet irradiation of DNA with isopropanol and di-tert-butylperoxide, causes a variety of base alterations. These include 8-(2-hydroxy-2-propyl)guanines, 8-(2-hydroxy-2-propyl)adenines and thymine dimers. An E. coli endonuclease against photoalkylated DNA was assayed by conversion of superhelical PM2 phage DNA to the nicked form. Enzyme activities were compared between extracts of strain BW9109 (xth-), lacking exonuclease III activity, and strain BW434 (xth-,nth-), deficient in both exonuclease III and endonuclease III. The endonuclease level in the double mutant against substrate photoalkylated DNA was under 20% of the activity in the mutant lacking only exonuclease III. Irradiation of the DNA substrate in the absence of isopropanol did not affect the activity in either strain. Analysis by polyacrylamide gel electrophoresis identified the sites of DNA cleavage by purified E. coli endonuclease III as cytosines, both in DNA irradiated at biologically significant wavelengths and in photoalkylated DNA. Neither 8-(2-hydroxy-2-propyl)purines, pyrimidine dimers, uracils nor 6-4'-(pyrimidin-2'-one)pyrimidines were substrates for the enzyme.     

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.     

T4 DNA polymerase (3''-5'') exonuclease, an enzyme for the detection and quantitation of stable DNA lesions: the ultraviolet light example.

Ultraviolet light irradiation of DNA results in the formation of two major types of photoproducts, cyclobutane dimers and 6-4' [pyrimidin-2'-one] -pyrimidine photoproducts. The enzyme T4 DNA polymerase possesses a 3' to 5' exonuclease activity and hydrolyzes both single and double stranded DNA in the absence of deoxynucleotide triphosphate substrates. Here we describe the use of T4 DNA polymerase associated exonuclease for the detection and quantitation of UV light-induced damage on both single and double stranded DNA. Hydrolysis of UV-irradiated single or double stranded DNA by the DNA polymerase associated exonuclease is quantitatively blocked by both cyclobutane dimers and (6-4) photoproducts. The enzyme terminates digestion of UV-irradiated DNA at the 3' pyrimidine of both cyclobutane dimers and (6-4) photoproducts. For a given photoproduct site, the induction of cyclobutane dimers was the same for both single and double stranded DNA. A similar relationship was also found for the induction of (6-4) photoproducts. These results suggest that the T4 DNA polymerase proofreading activity alone cannot remove these UV photoproducts present on DNA templates, but instead must function together with enzymes such as the T4 pyrimidine dimer-specific endonuclease in the repair of DNA photoproducts. The T4 DNA polymerase associated exonuclease should be useful for the analysis of a wide variety of bulky, stable DNA adducts.     

DNA polymerase β-dependent long patch base excision repair in living cells

We examined a role for DNA polymerase β (Pol β) in mammalian long patch base excision repair (LP BER). Although a role for Pol β is well known in single-nucleotide BER, information on this enzyme in the context of LP BER has been limited. To examine the question of Pol β involvement in LP BER, we made use of nucleotide excision repair-deficient human XPA cells expressing UVDE (XPA-UVDE), which introduces a nick directly 5′ to the cyclobutane pyrimidine dimer or 6-4 photoproduct, leaving ends with 3′-OH and 5′-phosphorylated UV lesion. We observed recruitment of GFP-fused Pol β to focal sites of nuclear UV irradiation, consistent with a role of Pol β in repair of UV-induced photoproducts adjacent to a strand break. This was the first evidence of Pol β recruitment in LP BER in vivo. In cell extract, a 5′-blocked oligodeoxynucleotide substrate containing a nicked 5′-cyclobutane pyrimidine dimer was repaired by Pol β-dependent LP BER. We also demonstrated Pol β involvement in LP BER by making use of mouse cells that are double null for XPA and Pol β. These results were extended by experiments with oligodeoxynucleotide substrates and purified human Pol β.     

Formation of cyclobutane thymine dimers photosensitized by pyridopsoralens: quantitative and qualitative distribution within DNA

As after irradiation with 254-nm UV light, exposure of thymidine and three isomeric pyridopsoralen derivatives to UVA radiation, in the dry state, leads to the formation of the six diastereomers of cyclobutadithymidine as the predominant reaction. This unexpected photosensitized reaction, which also gives rise to both 5R* and 5S* diastereomers of 5,6-dihydro-5-(alpha-thymidylyl)thymidine (or "spore" photoproduct), is selective since [2 + 2] dimerization of 2'-deoxycytidine was not detected under the same experimental conditions. The cis-syn isomer of cyclobutadithymine was also found to be produced within isolated DNA following UVA irradiation in aqueous solutions containing 7-methylpyrido[3,4-c]psoralen. Quantitatively, this photoproduct represents about one-fifth of the overall yield of the furan-side pyridopsoralen [2 + 2] photocycloadducts to thymine. DNA sequencing methodology was used to demonstrate that pyridopsoralen-photosensitized DNA is a substrate for T4 endonuclease V and Escherichia coli photoreactivating enzyme, two enzymes acting specifically on cyclobutane pyrimidine dimers. Furthermore, the dimerization reaction of thymine is sequence dependent, with a different specificity from that mediated by far-UV irradiation as inferred from gel sequencing experiments. Interestingly, adjacent thymine residues are excellent targets for 7-methylpyrido[3,4-c]psoralen-mediated formation of cyclobutadithymine in TTTTA and TTAAT sites, which are also the strongest sites for photoaddition. The formation of cyclobutane thymine dimers concomitant to that of thymine-furocoumarin photoadducts and their eventual implication in the photobiological effects of the pyridopsoralens are discussed.     

Restriction enzyme cleavage of ultraviolet-damaged simian virus 40 and pBR322 DNA

Cleavage of specific DNA sequences by the restriction enzymes EcoRI, HindIII and TaqI was prevented when the DNA was irradiated with ultraviolet light. Most of the effects were attributed to cyclobutane pyrimidine dimers in the recognition sequences; the effectiveness of irradiation was directly proportional to the number of potential dimer sites in the DNA. Combining EcoRI with dimer-specific endonuclease digestion revealed that pyrimidine dimers blocked cleavage within one base-pair on the strand opposite to the dimer but did not block cleavage three to four base-pairs away on the same strand. These are the probable limits for the range of influence of pyrimidine dimers along the DNA, at least for this enzyme. The effect of irradiation on cleavage by TaqI seemed far greater than expected for the cyclobutane dimer yield, possibly because of effects from photoproducts flanking the tetranucleotide recognition sequence and the effect of non-cyclobutane (6-4)pyrimidine photoproducts involving adjacent T and C bases.     

Sequence effect on alkali-sensitive sites in UV-irradiated SV40 DNA.

Ultraviolet light at 254 nm induces various kinds of DNA damage. We have located and quantified the pyrimidine (6-4) pyrimidone photoproducts along three hundred and forty two nucleotides of SV40 DNA. The level of photoproduct induction varies greatly according to the position on the DNA, but unlike what happens with pyrimidine dimers, the very adjacent nucleotides do not play a major role in the frequency of formation. A new alkali-sensitive site has been found on the ACA sequence after UV irradiation. This complex lesion is insensitive to the T4 endonuclease V and the E. coli photolyase, and may be involved with mutagenesis.     

The structure of the human AGT protein bound to DNA and its implications for damage detection

O6-Alklyguanine-DNA alkyltransferase (AGT) is an important DNA repair protein that protects cells from mutagenesis and toxicity arising from alkylating agents. We present an X-ray crystal structure of the wild-type human protein (hAGT) bound to double-stranded DNA with a chemically modified cytosine base. The protein binds at two different sites: one at the modified base, and the other across a sticky-ended DNA junction. The protein molecule that binds the modified cytosine base flips the base and recognizes it in its active site. The one that binds ends of neighboring DNA molecules partially flips an overhanging thymine base. This base is not inserted into the active-site pocket of the protein. These two different hAGT/DNA interactions observed in the structure suggest that hAGT may not detect DNA lesions by searching for the adduct itself, but rather for weakened and/or distorted base-pairs caused by base damage in the duplex DNA. We propose that hAGT imposes a strain on the DNA duplex and searches for DNA regions where the native structure is destabilized. The structure provides implications for pyrimidine recognition, improved inhibitor design, and a possible protein/protein interaction patch on hAGT.     

Dark Repair of Ultraviolet-Irradiated Deoxyribonucleic Acid by Bacteriophage T4: Purification and Characterization of a Dimer-Specific Phage-Induced Endonuclease

The purification and properties of an ultraviolet (UV) repair endonuclease are described. The enzyme is induced by infection of cells of Escherichia coli with phage T4 and is missing from extracts of cells infected with the UV-sensitive and excision-defective mutant T4V(1). The enzyme attacks UV-irradiated deoxyribonucleic acid (DNA) containing either hydroxymethylcytosine or cytosine, but does not affect native DNA. The specific substrate in UV-irradiated DNA appears to be pyrimidine dimer sites. The purified enzyme alone does not excise pyrimidine dimers from UV-irradiated DNA. However, dimer excision does occur in the presence of the purified endonuclease plus crude extract of cells infected with the mutant T4V(1).     

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.     

Active site plasticity of endonuclease V from Salmonella typhimurium

Base deamination is a major type of DNA damage under nitrosative stress. Endonuclease V initiates repair of deaminated base damage by making a nucleolytic incision one nucleotide away from the 3' side of the lesion. Within the endonuclease V family, the substrate specificities are different from one enzyme to another. In this study, we investigated deamination lesion cleavage activities of endonuclease V from the macrophage-residing pathogen, Salmonella typhimurium. Salmonella endonuclease V exhibits limited turnover on cleavage of deoxyinosine- and xanthosine-containing DNA. Binding analysis indicates that this single-turnover property is caused by tight binding to nicked products. The nicking activity is similar between the double-stranded deoxyinosine- and deoxyxanthosine-containing DNA. Cleavage rates are not affected by bases opposite the deoxyinosine or deoxyxanthosine lesions. The enzyme is also active on single-stranded deoxyinosine- and deoxyxanthosine-containing DNA. Unlike endonuclease V from Thermotoga maritima, Salmonella endonucleae V can only turnover deoxyuridine-containing DNA to a limited extent when substrate is in excess. Binding analysis indicates that Salmonella endonuclease V achieves tight binding to deoxyuridine-containing DNA, a property that distinguishes it from Thermotoga endonuclease V. Cleavage analysis on mismatch-containing DNA also indicates that the active site of Salmonella endonuclease V can accommodate pyrimidine-containing mismatches, resulting in more comparable cleavage of pyrimidine- and purine-containing mismatches. This comprehensive DNA cleavage and binding analysis reveals the plastic nature in the active site of Salmonella endonuclease V, which allows the enzyme to enfold both purine and pyrimidine deaminated lesions or base pair mismatches.