WAVELENGTH DEPENDENCE (150–290 nm) OF THE FORMATION OF THE CYCLOBUTANE DIMER AND THE (6–4) PHOTOPRODUCT OF THYMINE

The action cross sections for the formation of the cyclobutane dimer and the (6-4) photoproduct of thymine as well as the absorption cross sections of thymine were determined in the wavelength region between 150 and 290 nm. Thymine films sublimed on glass plates were irradiated by monochromatic photons in a vacuum; the induced photoproducts were quantitatively analyzed by high-performance liquid chromatography (HPLC). Under our conditions, two major peaks appeared on the HPLC chromatograms of irradiated samples. The two peaks were identified as being the cis-syn cyclobutane dimer and the (6-4) photoproduct, based on their HPLC retention times, absorption spectra in the effluent, and photochemical reactivity. The fractions of the two photoproducts increased linearly with the fluence at low fluences over the entire wavelength range. Their action cross sections were determined by the slopes of the linear fluence response curve at 10 nm intervals between 150 and 290 nm. The two action spectra showed a similar wavelength dependence and had a maximum at 270 nm as well as two minor peaks at 180 and 220 nm, at which wavelengths the peaks of the absorption spectrum of thymine sublimed on a CaF2 crystal plate appeared. The quantum yields had relatively constant values of around 0.008 for the dimer and 0.013 for the (6-4) photoproduct above 200 nm, decreasing to 0.003 and 0.006, respectively, at 150 nm as the wavelength became shorter.     

ESTABLISHMENT and CHARACTERIZATION OF A MONOCLONAL ANTIBODY RECOGNIZING THE DEWAR ISOMERS OF(6–4)PHOTOPRODUCTS

Abstract— We established a monoclonal antibody(DEM–1) that recognizes UV-induced DNA damage other than cyclobutane pyrimidine dimers or(6–4)photoproducts. The binding ofDEM–1 antibody to 254 nm UV-irradiated DNA increased with subsequent exposure to UV wavelengths longer than 310 nm, whereas that of the 64M-2 antibody specific for the(6–4)photoproduct decreased with this treatment. Furthermore, the increase inDEM–1 binding was inhibited by the presence of the 64M-2 antibody during the exposure. We concluded that theDEM–1 antibody specifically recognized the Dewar photoproduct, which is the isomeric form of the(6–4)photoproduct. TheDEM–1 antibody, however, also bound to DNA irradiated with high fluences of 254 nm UV, suggesting that 254 nm UV could induce Dewar photoproducts without subsequent exposure to longer wavelengths of UV. Furthermore, an action spectral study demonstrated that 254 nm was the most efficient wavelength for Dewar photoproduct induction in the region from 254 to 365 nm, as well as cyclobutane dimers and(6–4)photoproducts, although the action spectrum values in the U V-B region were significantly higher compared with those for cyclobutane dimer and(6–4)photoproduct induction.     

SIMULTANEOUS ESTABLISHMENT OF MONOCLONAL ANTIBODIES SPECIFIC FOR EITHER CYCLOBUTANE PYRIMIDINE DIMER OR (6-4)PHOTOPRODUCT FROM THE SAME MOUSE IMMUNIZED WITH ULTRAVIOLET-IRRADIATED DNA

Six new monoclonal antibodies (TDM-2, TDM-3, 64M-2, 64M-3, 64M-4 and 64M-5) specific for ultraviolet (UV) induced DNA damage have been established. In the antibody characterization experiments, two TDM antibodies were found to show a dose-dependent binding to UV-irradiated DNA (UV-DNA), decrease of binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, binding to DNA containing cyclobutane thymine dimers, and unchanged binding to UV-DNA after photoisomerization of (6-4)photoproducts to Dewar photoproducts. These results indicated that the epitope of TDM monoclonal antibodies was the cyclobutane pyrimidine dimer in DNA. On the other hand, four 64M antibodies were found to show a dose-dependent binding to UV-DNA, unchanged binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, undetectable binding to DNA containing thymine dimers, and decrease of binding to UV-DNA after photoisomerization of (6-4)photoproducts. These results indicated that the epitope of 64M antibodies was the (6-4)photoproduct in DNA. This is the first report of the simultaneous establishment of monoclonal antibodies against the two different types of photolesions from the same mouse. By using these monoclonal antibodies, we have succeeded in measuring both cyclobutane pyrimidine dimers and (6-4)photoproducts in the DNA from human primary cells irradiated with physiological UV doses.     

Far-UV-lnduced Dimeric Photoproducts in Short Oligonucleotides: Sequence Effects

Cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone adducts represent the two major classes of far-UV-induced DNA photoproducts. Because of the lack of appropriate detection methods for each individual photoproduct, little is known about the effect of the sequence on their formation. In the present work, the photoproduct distribution obtained upon exposure of a series of dinucleoside monophosphates to 254 nm light was determined. In the latter model compounds, the presence of a cytosine, located at either the 5′- or the 3′- side of a thymine moiety, led to the preferential formation of (6-4) adducts, whereas the cis-syn cyclobutane dimer was the main thymine-thymine photoproduct. In contrast, the yield of dimeric photoproducts, and particularly of (6-4) photoadducts, was very low upon irradiation of the cytosine–cytosine dinucleoside monophosphate. However, substitution of cytosine by uracil led to an increase in the yield of (6-4) photoproduct. It was also shown that the presence of a phosphate group at the 5′- end of a thymine-thymine dinucleoside monophosphate does not modify the photoproduct distribution. As an extension of the studies on dinucleoside monophosphates, the trinucleotide TpdCpT was used as a more relevant DNA model. The yields of formation of the thymine-cytosine and cytosine–thymine (6-4) photoproducts were in a 5:1 ratio, very close to the value obtained upon photolysis of the related dinucleoside monophosphates. The characterization of the two TpdCpT (6-4) adducts was based on H NMR, UV and mass spectroscopy analyses. Additional evidence for the structures was inferred from the analysis of the enzymatic digestion products of the (6-4) adducts of TpdCpT with phosphodiesterases. The latter enzymes were shown to induce the quantitative release of the photoproduct as a modified dinucleoside monophosphate in a highly sequence-specific manner.     

Ultraviolet Irradiation Produces Novel Endonuclease Ill-Sensitive Cytosine Photoproducts at Dipyrimidine Sites

Ultraviolet light irradiation of DNA in vitro and in vivo induces cyclobutane dimers, (6–4) pyrimidine-pyrimi-done photoproducts and a variety of minor products. Using a denned DNA fragment, we have identified two classes of sites that can be cleaved by Escherichia coli endonuclease III: single cytokines whose heat lability corresponds to that of cytosine hydrates and more heat-stable dipyrimidines containing cytosine. The dipyrimidine products are induced at sites suggestive of (6–4) photoproducts but are not recognized as (6–4) photoproducts by radioimmunoassay. Use of oligonucleotides containing a single cyclobutane thymine dimer, a (6–4) photoproduct or the Dewar photoisomer of the (6–4) photoproduct also indicated that these products are not substrates for endonuclease III. We have therefore identified a minor UV photoproduct that has the same sequence specificity as the two major dipyrimidine photoproducts; it may be a minor isomer, a unique derivative or an oxidative lesion confined to dipyrimidine sites. Its biological significance is not yet known but may be masked by the preponderance of major products at the same sites. Its occurrence at the particular site in dipyrimidine sequences involved in the mutagenic action of UV photoproducts suggests that it may play a role in generating C to T transitions that are common UV-induced mutations.     

Structural factors controlling the action of UV light in nucleic acids

Abstract— UV light induced conformational effects of different deoxyoligonucleotides and deoxypolynucleotides containing thymine and adenine residues are investigated by means of CD measurements and quantum yield calculations. UV-irradiation at the wavelengths 254 , 280 and 313 nm indicate that unsensitized irradiation at low doses leads to thymine photoproduct formation of non-cyclobutane type. In contrast to that irradiation at 313 nm in the presence of acetophenone causes different changes in the CD spectra due to the formation of thymine dimers of the cyclobutane type structure. Quantum yield calculations demonstrate a pronounced dependence of the photoproduct formation on the nucleotide sequence of the oligomers. Thus, clustering of thymine dimer formation can be neglected. Adenine photoproducts in the (A.T) containing oligomers are only formed at higher fluences. > 1.5 × 10⁴ J/m² and are biological less important events.     

ULTRAVIOLET LIGHT IRRADIATION OF DEFINED-SEQUENCE DNA UNDER CONDITIONS OF CHEMICAL PHOTOSENSITIZATION

Abstract— The formation of cyclobutane pyrimidine dimers and UV light-induced (6-4) products was examined under conditions of triplet state photosensitization. DNA fragments of defined sequence were irradiated with 313 nm light in the presence of either acetone qr silver ion. UV irradiation in the presence of both silver ion and acetone enhanced the formation of TT cyclobutane dimers, yet no (6-4) photoproducts were formed at appreciable levels. When photoproduct formation was also measured in pyrimidine dinucleotides, only cyclobutane dimers were formed when the dinucleotides were exposed to 313 nm light in the presence of photosensitizer. The relative distribution of each type of cyclobutane dimer formed was compared for DNA fragments that were irradiated with 254, 313, or 313 nm UV light in the presence of acetone. The dimer distribution for DNA irradiated with 254 and 313 nm UV light were very similar, whereas the distribution for DNA irradiated with 313 nm light in the presence of acetone favored TT dimers. Alkaline labile lesions at guanine sites were also seen when DNA was irradiated with 313 nm light in the presence of acetone.     

The relative cytotoxicity of (6-4) photoproducts and cyclobutane dimers in mammalian cells

Abstract— The significance of the pyrimidine(6-4)pyrimidone photoproduct in mammalian cell killing is considered. Photochemical data indicate that the(6–4) photoproduct is induced at a substantial frequency compared to the cyclobutane dimer and that the action spectra for the induction of both lesions are equivalent. The repair of(6–4) photoproducts in various normal and UV-hypcrsensitive mammalian cell lines, including several recently derived somatic cell hybrids and transformants, is presented. The sensitivity of these cells to ultraviolet irradiation correlates better with the capacity to repair(6–4) photoproducts than cyclobutane dimers. These data are used to support that idea that the(6–4) photoproduct is one of the major cytotoxic lesions induced in DNA by ultraviolet light.     

Repair of the main UV-induced thymine dimeric lesions within Arabidopsis thaliana DNA: evidence for the major involvement of photoreactivation pathways.

The UV-B induced formation of thymine cis-syn cyclobutane dimer and related (6-4) photoproduct was monitored within DNA of cultured cells and plants of Arabidopsis thaliana. This was achieved using a sensitive and accurate HPLC-tandem mass spectrometry assay. It was found that the cyclobutane pyrimidine dimer was formed in a ninefold higher yield than the (6-4) photoproduct. The removal of the lesions was then studied by incubating irradiated cells either in the darkness, under visible light or upon exposure to UV-A radiation. Dark repair of both cyclobutane dimers and (6-4) photoproducts was found to be very ineffective. In contrast, a rapid decrease in the level of photoproducts was observed when UV-B-irradiated cells were exposed to UV-A and, to a lesser extent, to visible light. The removal of (6-4) adducts was found to occur more efficiently. These results strongly suggest that repair of UV-induced photolesions in plants is mainly mediated by photolyases.     

Action spectra for survival and spore photoproduct formation of Bacillus subtilis irradiated with short-wavelength (200-300 nm) UV at atmospheric pressure and in vacuo.

Spores of Bacillus subtilis are approximately ten times less likely to survive UV light irradiation in a vacuum than under atmospheric conditions. Photoproduct formation was studied in spores irradiated under ultrahigh vacuum (UHV) conditions and in spores irradiated at atmospheric pressure. In addition to the "spore photoproduct" 5-thyminyl-5,6-dihydrothymine (TDHT), which is produced in response to irradiation at atmospheric pressure, two additional photoproducts, known as the cis-syn and trans-syn isomers of thymine dimer, are produced on irradiation in vacuo. The spectral efficiencies for photoproduct formation in spores are reduced under vacuum conditions compared with atmospheric conditions by a factor of 2-6, depending on the wavelength. Because formation of TDHT does not increase after irradiation in vacuo, TDHT cannot be responsible for the observed vacuum effect. Vacuum specific photoproducts may cause a synergistic response of spores to the simultaneous action of UV light and UHV. An increased quantum efficiency, destruction of repair systems and formation of irreparable lesions are postulated for the enhanced sensitivity of B. subtilis spores to UV radiation in vacuo.     

THE BIOLOGY OF THE (6–4) PHOTOPRODUCT

The (6-4) photoproduct is an important determinant of the lethal and mutagenic effects of UV irradiation of biological systems. The removal of this lesion appears to correlate closely with the early DNA repair responses of mammalian cells, including DNA incision events, repair synthesis and removal of replication blocks. The processing of (6-4) photoproducts and cyclobutane dimers appears to be enzymatically coupled in bacteria and most mammalian cell lines examined (i.e. a mutation affecting the repair of one lesion also often affects the other), although exceptions exist in which repair capacity may be evident for one photoproduct and not the other (e.g. UV61 and the XP revertant cell line). These differences in the processing of the two photoproducts in some cell lines of human and rodent origin suggest that in mammalian cells, different pathways for the repair of (6-4) photoproducts and cyclobutane dimers may be used. This observation is further supported by pleiotropic repair phenotypes such as those observed in CHO complementation class 2 mutants (e.g., UV5, UVL-1, UVL-13, and V-H1). Indirect data, from HCR of UV irradiated reported genes and the cytotoxic responses of UV61, suggest that the (6-4) photoproduct is cytotoxic in mammalian cells and may account for 20 to 30% of the cell killing after UV irradiation of rodent cells. Cytotoxicity of the (6-4) photoproduct may be important in the etiology of sunlight-induced carcinogenesis, affecting mutagenesis as well as tumorigenesis. The intricate photochemistry of the (6-4) photoproduct, its formation and photoisomerization, is in itself extremely interesting and may also be relevant to sunlight carcinogenesis. The data reviewed in this article support the notion that the (6-4) photoproduct and its Dewar photoisomer are important cytotoxic determinants of UV light. The idea that the (6-4) photoproduct is an important component in the spectrum of UV-induced cytotoxic damage may help clarify our understanding of why rodent cells survive the effects of UV irradiation as well as human cells, without apparent cyclobutane dimer repair in the bulk of their DNA. The preferential repair of cyclobutane dimers in essential genes has been proposed to account for this observation (Bohr et al., 1985, 1986; Mellon et al., 1986). The data reviewed here suggest that understanding the repair of a prominent type of noncyclobutane dimer damage, the (6-4) photoproduct, may also be important in resolving this paradox.     

PHOTOPHYSICS AND PHOTOCHEMISTRY OF PHOTOREACTIVATION

Abstract— Photoreactivating enzyme (PRE) monomerizes cyclobutyl pyrimidine dimers formed in DNA by UV light ( Λ < 300 nm). The enzyme requires near UV and visible wavelengths (300 < Λ < 600 nm) for activity. Possible mechanisms of action of the PRE are suggested by non-enzymatic processes in which pyrimidine dimers are monomerized by UV and visible light. Two such non-enzymatic processes are (a) photolysis of dimers resulting from direct absorption of UV, and (b) sensitized monomerization involving charge transfer complexes. Several lines of evidence suggest that the mechanism of action of the PRE more closely resembles (b) than (a). Recent experiments on the PRE from E. coli reveal the presence of new long wavelength absorption which may indicate the presence of a ground state complex. The known ability of PRE to monomerize dimers of thymine, cytosine and uracil suggests that the carbonyl groups at 2 position of the pyrimidine ring may be important in the interaction between enzyme and dimer.     

UV mutagenic photoproducts in Escherichia coli and human cells: a molecular genetics perspective on human skin cancer

Abstract— The relevance of photoproducts produced by 254 nm irradiation to human skin cancer is first critically evaluated. Experiments identifying the mutagenic photoproducts at 254 nm are then described. Mutations are primarily due to the(6–4) photoproduct and the cyclobutane pyrimidine dimer, both in E. coli and in human cells. The(6–4) photoproduct may be more important in E. coli and the cyclobutane dimer more important in mammalian cells. In human cells, mutations occur at the C of a TC, CT, or CC cyclobutane dimer, but not at TT cyclobutane dimers, and also appear to occur, less frequently, at the C of TC and CC(6–4) photoproducts. The local structure of DNA is more important in determining the frequency of mutation at a site than is the photoproduct frequency at that site. The effect of DNA structure appears to be due to site-specific lethality.     

INDUCTION OF PHOTOPRODUCTS IN SYNTHETIC POLYNUCLEOTIDES BY FAR AND NEAR ULTRAVIOLET RADIATION

Single and double-stranded polynucleotides of thymine and cytosine have been used to analyse the photoproducts produced by irradiation with far or near UV light. Reversed-phase high performance liquid chromatography was used to detect and quantify cyclobutane dimers and Pyr(6–4)Pyo adducts produced by 254 nm. At 320 nm 10-times less Thy(5–6)Thy dimers and one half the number of Cyt(5–6)Cyt dimers were induced; no Pyr(6–4)Pyo adducts were evident. HPLC has recently been applied to the isolation and characterization of various nucleic acid substituents and their UV-photoproducts. The relative retention times of pyrimidines and their UV photoproducts on HPLC reflect differences in the hydrophobicity of the compounds being separated. Hence, the more hydrophobic (less polar) a compound is, the greater its capacity to bind to the column and the greater its retention time; for T o T, C<>T and C<>C dimers this difference may result from variations in the number of methyl moieties (Cadet et al., 1983). The retention time of the compound also depends on its stereochemistry. Separation of the four stereoisomers of T<>T by HPLC shows that compounds which are molecular equivalents can be more or less accessible to the non-polar stationary phase depending on their conformation (Cadet, 1980). The relatively long retention times of the bipyrimidine photoadducts suggest a structural configuration which allows greater access to the hydrophobic moieties of the molecule. It is intriguing to consider that this difference in conformation may also be reflected in DNA–protein interactions such as binding by UV-endonucleases or antibodies directed against UV photodamage. Proteins can be used as sensitive probes for photoproduct induction and repair but an accurate evaluation of their specificity is required. It was the intent of this paper not only to compare the induction parameters for various dimers in pyrimidine homo-polymers but to provide controlled substrates which can be used to define the relative binding efficiencies of repair enzymes and antibodies for different types of photoproducts.     

Neutral histidine and photoinduced electron transfer in DNA photolyases

The two major UV-induced DNA lesions, the cyclobutane pyrimidine dimers (CPD) and (6-4) pyrimidine-pyrimidone photoproducts, can be repaired by the light-activated enzymes CPD and (6-4) photolyases, respectively. It is a long-standing question how the two classes of photolyases with alike molecular structure are capable of reversing the two chemically different DNA photoproducts. In both photolyases the repair reaction is initiated by photoinduced electron transfer from the hydroquinone-anion part of the flavin adenine dinucleotide (FADH(-)) cofactor to the photoproduct. Here, the state-of-the-art XMCQDPT2-CASSCF approach was employed to compute the excitation spectra of the respective active site models. It is found that protonation of His365 in the presence of the hydroquinone-anion electron donor causes spontaneous, as opposed to photoinduced, coupled proton and electron transfer to the (6-4) photoproduct. The resulting neutralized biradical, containing the neutral semiquinone and the N3'-protonated (6-4) photoproduct neutral radical, corresponds to the lowest energy electronic ground-state minimum. The high electron affinity of the N3'-protonated (6-4) photoproduct underlines this finding. Thus, it is anticipated that the (6-4) photoproduct repair is assisted by His365 in its neutral form, which is in contrast to the repair mechanisms proposed in the literature. The repair via hydroxyl group transfer assisted by neutral His365 is considered. The repair involves the 5'base radical anion of the (6-4) photoproduct which in terms of electronic structure is similar to the CPD radical anion. A unified model of the CPD and (6-4) photoproduct repair is proposed.     

THE ACTION SPECTRA FOR UV-INDUCED SUPPRESSION OF MLR and MECLR SHOW THAT IMMUNOSUPPRESSION IS MEDIATED BY DNA DAMAGE

-Ultraviolet-B (UVB,280–320 nm) radiation can promote the induction of skin cancer by two mechanisms: damage of epidermal DNA and suppression of the immune system, allowing the developing tumor to escape immune surveillance. The mixed lymphocyte reaction (MLR) and the mixed epidermal cell lymphocyte reaction (MECLR) are commonly used methods to study the immunosuppressive effects of UVB radiation. To obtain a better understanding of the mechanism by which UVB radiation decreases the alloactivating capacity of in vitro-irradiated cells, action spectra for the MLR and MECLR were determined. Suspensions of peripheral blood mononuclear cells or epidermal cells were irradiated with monochromatic light of 254, 297, 302 or 312 nm and used as stimulator cells in the MLR or MECLR. Using dose-response curves for each wavelength, the action spectra were calculated. Both MLR and MECLR action spectra had a maximum at 254 nm and a relative sensitivity at 312 nm that was a thousand times lower than at 254 nm. Strikingly, the action spectra corresponded very closely to the action spectra that were found by Matsunaga et al. (Photochem. Photobiol. 54,403–410, 1991) for the induction of thymine dimers and (6-4)photoproducts in irradiated calf thymus DNA solutions, strongly suggesting that the UV-induced abrogation of the MLR and MECLR responses is mediated by UV-induced DNA damage. Furthermore, the action spectra for the MLR and MECLR were similar, suggesting that they share a common mechanism for UV-induced suppression.     

INDUCTION OF CHROMOSOME ABERRATIONS IN ICR 2A FROG CELLS EXPOSED TO265–313 nm MONOCHROMATIC ULTRAVIOLET WAVELENGTHS and PHOTOREACTIVATING LIGHT

Abstract— Exposure of ICR 2A cells to either 265, 289, 302 or 313 nm monochromatic UV wavelengths caused the induction of chromosome aberrations with chromatid gaps and breaks being the most common type of aberration detected. Treatment of U V-irradiated cells with photoreactivating light (PRL) resulted in a lower yield of aberrations demonstrating that pyrimidine dimers are involved in the formation of chromosome aberrations induced by the UV wavelengths tested. However, the decrease in the level of aberrations resulting from PRL treatment of 313 nm-irradiated cells was significantly less than for the other wavelengths indicating that non-dimer photoproducts may have played an important additional role in the induction of chromosome aberrations by this UV wavelength.     

INDUCTION OF DIRECT AND INDIRECT SINGLE-STRAND BREAKS IN HUMAN CELL DNA BY FAR- AND NEAR-ULTRAVIOLET RADIATIONS: ACTION SPECTRUM AND MECHANISMS

Abstract— An action spectrum for the immediate induction in DNA of single-strand breaks (SSBs, frank breaks plus alkali-labile sites) in human P3 teratoma cells in culture by monochromatic 254-, 270-, 290-, 313-, 334-, 365-, and 405-nm radiation is described. The cells were held at +0.5C during irradiation and were Iysed immediately for alkaline sedimentation analysis following the irradiation treatments. Linear fluence responses were observed over the fluence ranges studied for all energies. Irradiation of the cells in a D₂O environment (compared with the normal H₂O environment) did not alter the rate of induction of SSBs by 290-nm radiation, whereas the D₂O environment enhanced the induction of SSBs by 365- and 405-nm irradiation. Analysis of the relative efficiencies for the induction of SSBs, corrected for quantum efficiency and cellular shielding, revealed a spectrum that coincided closely with nucleic acid absorption below 313 nm. At longer wavelengths, the plot of relative efficiency vs . wavelength contained a minor shoulder in the same wavelength region as that observed in a previously obtained action spectrum for stationary phase Bacillus subtilis cells. Far-UV radiation induced few breaks relative to pyrimidine dimers, whereas in the near-UV region of radiation, SSBs account for a significant proportion of the lesions relative to dimers, with a maximum number of SSBs per lethal event occurring at 365-nm radiation.     

PYRIMIDINE DIMERS INDUCED IN ESCHERICHIA COLI DNA BY ULTRAVIOLET RADIATION PRESENT IN SUNLIGHT

Abstract— Escherichia coli DNA was irradiated with various wavelengths of monochromatic UV light from 254 to 320 nm, and the relative yields of the different cyclobutane pyrimidine dimers determined. Cytosine–thymine dimers (C < > T) were more frequent than thymine dimers (T < > T) at low fluences of 300 and 313 nm light, whereas the reverse was true at either longer or shorter wavelengths. Thus, in the solar UV range deemed responsible for skin cancer (i.e. 295–315 nm), C < > T are probably more important than T < > T.     

CYCLOBUTANE DIMERS AND (6-4)PHOTOPRODUCTS IN HUMAN CELLS ARE MENDED WITH THE SAME PATCH SIZES

The size of excision repair patches corresponding to excision of (6-4) pyrimidine-pyrimidone photoproducts and (5-5, 6-6) cyclobutane dimers have been independently determined by using bromodeoxyuridine substitution and density increases in isopycnic gradients of small DNA fragments. The two classes of photoproducts were distinguished by using (a) a xeroderma pigmentosum (XP) revertant cell line that excises (6-4) photoproducts normally, but does not excise cyclobutane dimers from bulk DNA or from an actively transcribed sequence; (b) an XP cell line containing the denV gene of bacteriophage T4, which repairs only cyclobutane dimers by a unique glycosylase mechanism, and (c) normal cells analyzed during time intervals in which cyclobutane dimer repair is the main repair process in action. The patch sizes for the two lesions were similar under all conditions and were estimated to be approximately 30-40 bases. These values are slightly large than corresponding estimates for Escherichia coli and Saccharomyces cerevisiae but close to estimates from in vitro experiments with human cell extracts. The size of 30 bases may consequently be very close to the actual distance between cleavage sites made on either side of a photoproduct during repair.