Escherichia coli DNA photolyase reverses cyclobutane pyrimidine dimers but not pyrimidine-pyrimidone (6-4) photoproducts

The effect of purified Escherichia coli DNA photolyase on the UV light-induced pyrimidine-pyrimidone (6-4) photoproduct and cyclobutane pyrimidine dimer was investigated in vitro using enzyme purified from cells carrying the cloned phr gene (map position, 15.7 min). Photoproducts were examined both as site-specific lesions in end-labeled DNA and as chromatographically identified products in uniformly labeled DNA. E. coli DNA photolyase removed cyclobutane dimers but had no activity on pyrimidine-pyrimidone (6-4) photoproducts. Photoreactivation can therefore be used to separate the biological effects of these two UV light-induced molecular lesions.     

UV light-induced DNA damage and tolerance for the survival of nucleotide excision repair-deficient human cells

DNA damage can cause cell death unless it is either repaired or tolerated. The precise contributions of repair and tolerance mechanisms to cell survival have not been previously evaluated. Here we have analyzed the cell killing effect of the two major UV light-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs), in nucleotide excision repair-deficient human cells by expressing photolyase(s) for light-dependent photorepair of either or both lesions. Immediate repair of the less abundant 6-4PPs enhances the survival rate to a similar extent as the immediate repair of CPDs, indicating that a single 6-4PP lesion is severalfold more toxic than a CPD in the cells. Because UV light-induced DNA damage is not repaired at all in nucleotide excision repair-deficient cells, proliferation of these cells after UV light irradiation must be achieved by tolerance of the damage at replication. We found that RNA interference designed to suppress polymerase zeta activity made the cells more sensitive to UV light. This increase in sensitivity was prevented by photorepair of 6-4PPs but not by photorepair of CPDs, indicating that polymerase zeta is involved in the tolerance of 6-4PPs in human cells.     

Substrate specificity of ultraviolet DNA endonuclease (UVDE/Uve1p) from Schizosaccharomyces pombe.

Schizosaccharomyces pombe ultraviolet DNA endonuclease (UVDE or Uve1p) has been shown to cleave 5' to UV light-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PP). This endonuclease is believed to function in the initial step in an alternative excision repair pathway for the removal of DNA damage caused by exposure to UV light. An active truncated form of this protein, Delta228-Uve1p, has been successfully overexpressed, affinity purified and partially characterized. In the present study we present data from a detailed substrate specificity trial. We have determined that the substrate range of Uve1p is much greater than was originally believed. We demonstrate that this DNA damage repair protein is capable of recognizing an array of UV-induced DNA photoproducts (cis-syn-, trans-syn I- and trans-syn II CPDs, 6-4PP and Dewar isomers) that cause varying degrees of distortion in a duplex DNA molecule. We also demonstrate that Uve1p recognizes non-UV-induced DNA damage, such as platinum-DNA GG diadducts, uracil, dihydrouracil and abasic sites. This is the first time that a single DNA repair endonuclease with the ability to recognize such a diverse range of lesions has been described. This study suggests that Uve1p and the alternative excision repair pathway may participate broadly in the repair of DNA damage.     

Nuclease SP: a novel enzyme from spinach that incises damaged duplex DNA preferentially at sites of adenine.

A novel endonuclease has been isolated from extracts of spinach leaves (Spinacia oleracea). The enzyme has been purified by a series of column chromatography steps and has a molecular size of approximately 43,000 daltons. The spinach endonuclease cleaved double stranded DNA damaged by ultraviolet light or cis-diamminedichloroplatinum (II) primarily at sites of adenine when end-labelled DNA fragments of defined sequence were employed as substrates. The nature of the structural distortion contained in damaged, duplex DNA appears to be an important determinant for endonuclease cleavage. DNA helical distortions produced by UV light-induced (6-4) pyrimidine-pyrimidone photoproducts, but not cyclobutane pyrimidine dimers are recognized by the enzyme. The DNA cleavage products generated by the enzyme contain 3'-hydroxyl and 5'-phosphoryl termini. Single stranded DNA and RNA are hydrolyzed by the spinach endonuclease. This enzyme, which we call nuclease SP, is similar in several respects to other single-strand-specific nucleases such as N. crassa and mung bean nucleases and may function in DNA repair and/or recombination events in spinach cells. Nuclease SP should be a useful tool for the analysis of (6-4) photoproducts occurring in duplex DNA.     

Immunoprecipitation of pyrimidine(6-4)pyrimidone photoproducts and cyclobutane pyrimidine dimers in uv-irradiated DNA

Biological studies suggest that a significant proportion of the cytotoxicity observed in mammalian cells after uv irradiation may be due to damage other than cyclobutane dimers in DNA. Although pyrimidine-pyrimidone (6-4) photoproducts have been implicated as major contributors to cell lethality, their induction has been measured at considerably less than cyclobutane pyrimidine dimers when measured by chromatographic techniques. Because the yield of (6-4) photoproducts may be reduced by their lability to extreme heat and pH, we have advised an alternative, immunological quantification which does not require DNA hydrolysis. Affinity-purified rabbit antisera were used to precipitate low molecular weight 32P-labeled PM2 DNA irradiated with increasing fluences of uv light. DNA of known molecular weight was used to determine rates of induction for antibody-binding sites associated with (6-4) photoproducts and cyclobutane dimers. These rates were calculated to be 0.6 (6-4) photoproducts and 1.2 cyclobutane dimers/10(8) Da/J/m2. At low uv fluences (6-4) photoproducts were induced at one-half the rate of cyclobutane dimers, whereas at higher fluences (6-4) photoproducts predominated.     

Nonrandom induction of pyrimidine-pyrimidone (6-4) photoproducts in ultraviolet-irradiated human chromatin

Radioimmunoassays that detect pyrimidine-pyrimidone (6-4) photoproducts and cyclobutane dimers were used to determine the relative induction of these photoproducts in nucleosomal (core) and internucleosomal (linker) DNA in human cell chromatin irradiated with UV light. Cyclobutane dimers were formed in equal amounts/nucleotide in core and linker DNA, whereas (6-4) photoproducts occurred with 6-fold greater frequency/nucleotide in linker DNA.     

Crystal structure of the 64M-2 antibody Fab fragment in complex with a DNA dT(6-4)T photoproduct formed by ultraviolet radiation

DNA photoproducts with (6-4) pyrimidine-pyrimidone adducts formed by ultraviolet radiation are implicated in mutagenesis and cancer, particularly skin cancer. The crystal structure of the Fab fragment of the murine 64M-2 antibody specific to DNA T(6-4)T photoproducts is determined as a complex with dT(6-4)T, a (6-4) pyrimidine-pyrimidone photodimer of dTpT, at 2.4 A resolution to a crystallographic R-factor of 0.199 and an R(free) value of 0.279. The 64M-2 Fab molecule is in an extended arrangement with an elbow angle of 174 degrees, and its five complementarity-determining regions, except L2, are involved in the ligand binding. The bound dT(6-4)T ligand adopting a ring structure with (6-4) linked 5' thymine-3' pyrimidone bases is fully accommodated in an antigen-binding pocket of about 15 Ax10 A. The 5'-thymine and 3'-pyrimidone bases are in half-chair and planar conformations, respectively, and are nearly perpendicular to each other. The 5'-thymine base is hydrogen-bonded to Arg95H and Ser96H, and is in van der Waals contact with Tyr100iH. The 3'-pyrimidone base is hydrogen-bonded to His35H, and is in contact with Trp33H. Three water molecules are located at the interface between the bases and the Fab residues. Hydrogen bonds involving these water molecules also contribute to Fab recognition of the dT(6-4)T bases. The sugar-phosphate backbone connecting the bases is surrounded by residues His27dL, Tyr32L, Ser92L, Trp33H, and Ser58H, but is not hydrogen-bonded to these residues.     

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.     

Base sequence specificity of a monoclonal antibody binding to (6-4)photoproducts.

The DNA base sequence specificity of the 64M-1 monoclonal antibody, which recognizes ultraviolet (UV)-induced (6-4)photoproducts, was characterized. The 64M-1 antibody strongly bound to UV-poly(dU) as well as to UV-poly(dT), and weakly to UV-poly(dC), UV-poly(me5dC) and UV-poly(rU). A competitive inhibition assay using UV-oligo(dT)8, UV-oligo(dTdC)4, UV-oligo(dC)8, UV-PvuI linker (GCGATCGC) and UV-PvuII linker (GCAGCTGC) indicated that the main (6-4)photoproducts detected by the 64M-1 antibody in UV-irradiated DNA are TT(6-4)photoproducts and TC(6-4)photoproducts. Comparison between dTpdT(6-4)photoproduct and dTpdC(6-4)photoproduct showed that the affinity of the 64M-1 antibody for dTpdT(6-4)photoproduct was about 5 times higher than that for dTpdC(6-4)photoproduct. The antibody also binds to isolated TT(6-4)photoproducts.     

Identification and characterization of an intermediate in the alkali degradation of (6-4) photoproduct-containing DNA

The (6-4) photoproduct formed by ultraviolet light is known as an alkali-labile DNA lesion. Strand breaks occur at (6-4) photoproducts when UV-irradiated DNA is treated with hot alkali. We have analyzed the degradation reaction of this photoproduct under alkaline conditions using synthetic oligonucleotides. A tetramer, d(GT(6-4)TC), was prepared, and its degradation in 50 mm KOH at 60 degrees C was monitored by high performance liquid chromatography. A single peak with a UV absorption spectrum similar to that of the starting material was detected after the reaction, and this compound was regarded as an intermediate before the strand break. The formation of this intermediate was compared with intermediates from the degradation of other alkali-labile lesions such as the abasic site, thymine glycol, and 5,6-dihydrothymine. The results strongly suggested that the first step of the alkali degradation of the (6-4) photoproduct was the hydrolysis between the N3 and C4 positions of the 5'-pyrimidine component. Analyses by NMR spectroscopy and mass spectrometry supported the chemical structure of this product. Assays of the complex formation with XPC.HR23B and the translesion synthesis by DNA polymerase eta revealed that the biochemical properties are indistinguishable between the intact and hydrolyzed photoproducts.     

A new ATP-independent DNA endonuclease from Schizosaccharomyces pombe that recognizes cyclobutane pyrimidine dimers and 6-4 photoproducts.

We have discovered a new DNA endonuclease in the fission yeast Schizosaccharomyces pombe which recognizes cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone photoproducts. S. pombe DNA endonuclease (SPDE) catalyzes a single ATP-independent incision immediately 5' to the UV photoproduct and generates termini containing 3' hydroxyl and 5' phosphoryl groups. Based on these properties, we propose that SPDE may function in a DNA repair capacity, representing the initial recognition/cleavage step of a DNA excision repair pathway.     

Mammalian polymerase ζ is essential for post-replication repair of UV-induced DNA lesions

DNA polymerase ζ is believed to be an essential constituent of DNA damage tolerance, comprising several pathways that allow the replication of DNA templates containing unrepaired damage. We wanted to better define the role of polymerase ζ in DNA damage tolerance in mammalian cells. To this aim we have investigated replication of ultraviolet light-damaged DNA templates in mouse embryonic fibroblasts deficient for Rev3, the catalytic subunit of polymerase ζ. We found that Rev3 is important for a post-replication repair pathway of helix-distorting [6-4]pyrimidine-pyrimidone photoproducts and, to a lesser extent, of cyclobutane pyrimidine dimers. Unlike its partner Rev1, Rev3 appears not to be involved in an immediate translesion synthesis pathway at a stalled replication fork. The deficiency of Rev3^?/? MEFs in post-replication repair of different photoproducts contributes to the extreme sensitivity of these cells to UV light.     

Individual determination of the yield of the main UV-induced dimeric pyrimidine photoproducts in DNA suggests a high mutagenicity of CC photolesions

Bipyrimidine photoproducts induced in DNA by UVB radiation include cyclobutane dimers, (6-4) photoproducts, and their related Dewar valence isomers. Even though these lesions have been extensively studied, their rate of formation within DNA is still not known for each possible bipyrimidine site (TT, TC, CT, and CC). Using a method based on the coupling of liquid chromatography to mass spectrometry, we determined the distribution of the 12 possible bipyrimidine photoproducts within isolated and cellular DNA. TT and TC were found to be the most photoreactive sequences, whereas lower amounts of damage were produced at CT and CC sites. In addition to this quantitative aspect, sequence effects were observed on the relative yield of (6-4) adducts with respect to cyclobutane pyrimidine dimers. Another interesting result is the lack of formation of Dewar valence isomers in detectable amounts within the DNA of cells exposed to low doses of UVB radiation. The photoproduct distribution obtained does not fully correlate with the UV mutation spectrum. A major striking observation deals with the low yield of cytosine-cytosine photoproducts which are likely to be associated with the UV-specific CC to TT tandem mutation.     

The induction and repair of lesions produced by the photolysis of (6-4) photoproducts in normal and UV-hypersensitive human cells

A radioimmunoassay was used to study the induction and repair of damage produced by the photolysis of (6-4) photoproducts in normal and UV-sensitive human cells. Photochemical conditions were established to optimize the production of photolyzed (6-4) photoproducts in human cell DNA with minimal induction of other photoproducts. The repair of this photoproduct, presumed to be a Dewar pyrimidinone, was similar to that determined for the (6-4) photoproduct, with most of the antibody-binding sites removed within 4 h post-photolysis. Whereas xeroderma pigmentosum group A cells were deficient in the repair of this lesion, an XP variant and two cell lines selectively hypersensitive to UVB-irradiation were shown to have normal repair. The radioimmunoassay was further used to demonstrate the alkali-lability of the (6-4) photolysis product.     

UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells.

We used a simian virus 40-based shuttle vector plasmid, pZ189, to determine the role of pyrimidine cyclobutane dimers in UV light-induced mutagenesis in monkey cells. The vector DNA was UV irradiated and then introduced into monkey cells by transfection. After replication, vector DNA was recovered from the cells and tested for mutations in its supF suppressor tRNA marker gene by transformation of Escherichia coli carrying a nonsense mutation in the beta-galactosidase gene. When the irradiated vector was treated with E. coli photolyase prior to transfection, pyrimidine cyclobutane dimers were removed selectively. Removal of approximately 90% of the pyrimidine cyclobutane dimers increased the biological activity of the vector by 75% and reduced its mutation frequency by 80%. Sequence analysis of 72 mutants recovered indicated that there were significantly fewer tandem double-base changes and G X C----A X T transitions (particularly at CC sites) after photoreactivation of the DNA. UV-induced photoproducts remained (although at greatly reduced levels) at all pyr-pyr sites after photoreactivation, but there was a relative increase in photoproducts at CC and TC sites and a relative decrease at TT and CT sites, presumably due to a persistence of (6-4) photoproducts at some CC and TC sites. These observations are consistent with the fact that mutations were found after photoreactivation at many sites at which only cyclobutane dimers would be expected to occur. From these results we conclude that UV-induced pyrimidine cyclobutane dimers are mutagenic in DNA replicated in monkey cells.     

Utilization of DNA photolyase, pyrimidine dimer endonucleases, and alkali hydrolysis in the analysis of aberrant ABC excinuclease incisions adjacent to UV-induced DNA photoproducts.

ABC excinuclease of Escherichia coli removes 6-4 photoproducts and pyrimidine dimers from DNA by making two single strand incisions, one 8 phosphodiester bonds 5' and another 4 or 5 phosphodiester bonds 3' to the lesion. We describe in this communication a method, which utilizes DNA photolyase from E. coli, pyrimidine dimer endonucleases from M. luteus and bacteriophage T4, and alkali hydrolysis, for analyzing the ABC excinuclease incision pattern corresponding to each of these photoproducts in a DNA fragment. On occasion, ABC excinuclease does not incise DNA exclusively 8 phosphodiester bonds 5' or 4 or 5 phosphodiester bonds 3' to the photoproduct. Both the nature of the adduct (6-4 photoproduct or pyrimidine dimer) and the sequence of neighboring nucleotides influence the incision pattern of ABC excinuclease. We show directly that photolyase stimulates the removal of pyrimidine dimers (but not 6-4 photoproducts) by the excinuclease. Also, photolyase does not repair CC pyrimidine dimers efficiently while it does repair TT or TC pyrimidine dimers.     

Poly(ADP-ribose) Contributes to an Association between Poly(ADP-ribose) Polymerase-1 and Xeroderma Pigmentosum Complementation Group A in Nucleotide Excision Repair

Exposure to ultraviolet radiation (UVR) promotes the formation of UVR-induced, DNA helix distorting photolesions such as (6-4) pyrimidine-pyrimidone photoproducts and cyclobutane pyrimidine dimers. Effective repair of such lesions by the nucleotide excision repair (NER) pathway is required to prevent DNA mutations and chromosome aberrations. Poly(ADP-ribose) polymerase-1 (PARP-1) is a zinc finger protein with well documented involvement in base excision repair. PARP-1 is activated in response to DNA damage and catalyzes the formation of poly(ADP-ribose) subunits that assist in the assembly of DNA repair proteins at sites of damage. In this study, we present evidence for PARP-1 contributions to NER, extending the knowledge of PARP-1 function in DNA repair beyond the established role in base excision repair. Silencing the PARP-1 protein or inhibiting PARP activity leads to retention of UVR-induced photolesions. PARP activation following UVR exposure promotes association between PARP-1 and XPA, a central protein in NER. Administration of PARP inhibitors confirms that poly(ADP-ribose) facilitates PARP-1 association with XPA in whole cell extracts, in isolated chromatin complexes, and in vitro. Furthermore, inhibition of PARP activity decreases UVR-stimulated XPA chromatin association, illustrating that these relationships occur in a meaningful context for NER. These results provide a mechanistic link for PARP activity in the repair of UVR-induced photoproducts.     

An ultraviolet light-damaged DNA recognition protein absent in xeroderma pigmentosum group E cells binds selectively to pyrimidine (6-4) pyrimidone photoproducts.

The binding specificity was defined of a human ultraviolet light-damaged DNA recognition protein (UV-DRP), the activity of which is absent in some xeroderma pigmentosum complementation group E cells. Our results suggest that cyclobutane pyrimidine dimers (CPDs) are not high affinity UV-DRP binding sites--a finding consistent with other reports on this protein (Hirschfeld et al., (1990) Mol. Cell Biol., 10, 2041-2048). A major role for 6-4 photoproducts in UV-DRP binding was suggested in studies showing that irradiated oligonucleotides containing a T4C UV box sequence, which efficiently forms a TC 6-4 photoproduct, was a superior substrate for the UV-DRP when compared to a similar irradiated oligonucleotide having a T5 sequence. The latter sequence forms CPDs at a much higher frequency than 6-4 photoproducts. In a more direct approach, T4C-containing oligonucleotides complexed with the UV-DRP were separated from the unbound oligonucleotide fraction and the frequencies of 6-4 photoproducts in the two DNA populations were compared. The UV-DRP-bound fraction was highly enriched for the 6-4 lesion over the unbound fraction supporting the conclusion that 6-4 photoproducts are the principal binding cues for the UV-DRP.     

In vivo formation and repair of cyclobutane pyrimidine dimers and 6-4 photoproducts measured at the gene and nucleotide level in Escherichia coli

In vivo formation and repair of the major UV-induced DNA photoproducts, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4 PPs), have been examined at the gene and nucleotide level in Escherichia coli. Each type of DNA photoproduct has individually been studied using photoreactivation and two newly developed assays; the multiplex QPCR assay for damage detection at the gene level and the reiterative primer extension (PE) assay for damage detection at the nucleotide level. In the E. coli lacI and lacZ genes, CPDs and 6-4 PPs form in a 2:1 ratio, respectively, during UV irradiation. Repair of 6-4 PPs is more efficient than repair of CPDs since, on the average, 42% of 6-4 PPs are repaired in both genes in the first 40 min following 200 J/m² UV irradiation, while 1% of CPDs are repaired. The location, relative frequency of formation, and efficiency of repair of each type of photoproduct was examined in the first 52 codons of the E. coli lacI gene at the nucleotide level. Hotspots of formation were found for each type of lesion. Most photoproducts are at sites where both CPDs and 6-4 PPs are formed. Allowing 40 min of recovery following 200 J/m² shows that in vivo repair of 6-4 PPs is about fourfold more efficient than the repair of CPDs. Comparison of the lesion-specific photoproduct distribution of the lacI gene with a UV-induced mutation spectrum from wild-type cells shows that most mutational hotspots are correlated with sites of a majority of CPD formation. However, 6-4 PPs are also formed at some of these sites with relatively high frequency. This information, taken together with the observation that 6-4 PPs are repaired faster than CPDs, suggest that the cause of mutagenic hotspots in wild-type E. coli is inefficient repair of CPDs.