Specific recognition of single-stranded nucleic acids. Interaction of tryptophan-containing peptides with native, denatured, and ultraviolet-irradiated DNA

The binding of the tripeptide Lys-Trp-Lys to native, denatured, and ultraviolet-irradiated DNAs has been investigated by fluorescence spectroscopy. Two types of complexes are formed which both involve electrostatic interactions. Only one of them involves a stacking of the tryptophyl ring with nucleic acid bases. Quantitative analysis of fluorescence data shows that this stacking interaction is strongly favored in denatured as compared to native DNA. In ultraviolet-irradiated DNA, the peptide Lys-Trp-Lys binds selectively to unpaired regions around thymine dimers. Due to the stacking interaction of the aromatic amino acid with nucleic acid bases, this simple tripeptide is therefore able to discriminate between single-stranded and double-stranded regions in a nucleic acid.     

A maximum of two tryptophan residues in gene-32 protein from phage T4 undergo stacking interactions with single-stranded polynucleotides

The effect of specific photochemical and radiochemical modification of tryptophyl and cysteinyl residues of the gene 32 protein (gp 32) of bacteriophage T4 on its affinity towards single-stranded polynucleotides has been investigated. Oxidation of Cys residues of gp 32 by the free-radical anion I-.2 induces a partial loss of the protein affinity, probably by affecting the metal-binding domain which includes three of the four cysteine residues of gp 32. Ultraviolet irradiation of gp 32 in the presence of trichloroethanol results in the modification of three of its five Trp residues and total loss of the protein binding. Analysis of the relative affinity of ultraviolet-irradiated gp 32 for single-stranded polynucleotides suggest that modification of a Trp of enhanced reactivity occurs first and has no effect on the protein binding. Radiochemical modification of three Trp residues of gp 32 by (SCN)-.2 results in total loss of activity. Complexation of gp 32 with denatured DNA prior to gamma-irradiation protects two Trp residues and prevents the protein inactivation. These results suggest that at most two Trp residues are involved in stacking interactions with nucleic acid bases. However, time-resolved spectroscopic methods which allow us to monitor selectively the stacked tryptophan residues have not yielded evidence of more than a single residue undergoing such interactions.     

Characterization of a wide range base-damage-endonuclease activity of mammalian rpS3

Mammalian rpS3, a ribosomal protein S3 with a DNA repair endonuclease activity, nicks heavily UV-irradiated DNA and DNA containing AP sites. RpS3 calls for a novel endonucleolytic activity on AP sites generated from pyrimidine dimers by T4 pyrimidine dimer glycosylase activity. This study revealed that rpS3 cleaves the lesions including AP sites, thymine glycols, and other UV damaged lesions such as pyrimidine dimers. This enzyme does not have a glycosylase activity as predicted from its amino acid sequence. However, it has an endonuclease activity on DNA containing thymine glycol, which is exactly overlapped with UV-irradiated or AP DNAs, indicating that rpS3 cleaves phosphodiester bonds of DNAs containing altered bases with broad specificity acting as a base-damage-endonuclease. RpS3 cleaves supercoiled UV damaged DNA more efficiently than the relaxed counterpart, and the endonuclease activity of rpS3 was inhibited by MgCl2 on AP DNA but not on UV-irradiated DNA.     

Establishment of a monoclonal antibody recognizing cyclobutane-type thymine dimers in DNA: a comparative study with 64M-1 antibody specific for (6-4)photoproducts

We obtained a monoclonal antibody (TDM-1) binding to 313-nm UV-irradiated DNA in the presence of acetophenone. The binding of TDM-1 to 254-nm UV-irradiated DNA was not reduced with the subsequent irradiation of 313-nm UV. Furthermore, the treatment of UV-irradiated DNA with photolyase from E. coli and visible light exposure reduced both the antibody binding and the amount of thymine dimers in the DNA. A competitive inhibition assay revealed that the binding of TDM-1 to UV-irradiated DNA was inhibited with photolyase, but not with 64M-1 antibody specific for (6-4)photoproducts. These results suggest that TDM-1 antibody recognizes cyclobutane-type thymine dimers in DNA. Using TDM-1 and 64M-1 antibodies, we differentially measured each type of damage in DNA extracted from UV-irradiated mammalian cells. Repair experiments confirm that thymine dimers are excised from UV-irradiated cellular DNA more slowly than (6-4)photoproducts, and that the excision rates of thymine dimers and (6-4)photoproducts are lower in mouse NIH3T3 cells than in human cells.     

Inhibition of DNA polymerase activity by thymine hydrates.

Ultraviolet irradiation of DNA results in various pyrimidine modifications. We have demonstrated formation of both cis-thymine hydrate and trans-thymine hydrate (6-hydroxy-5,6-dihydrothymine) in UV-irradiated poly(dA-dT):poly(dA-dT). Both are released from DNA as free bases by bacterial and human glycosylases. Thymine hydrates are stable in DNA and can be detected in control, unirradiated substrates. We examined the effects of thymine hydrates in UV-irradiated substrate poly(dA-dT):poly(dA-dT) on E. coli DNA polymerase I activity. Enzymic incorporation of labeled thymidine-5'-monophosphate significantly decreased with increasing UV dose. Reversal of DNA thymine hydrates to thymines by mild heating of the substrate prior to enzymic reaction resulted in partial recovery of nucleotide incorporation. Cyclobutane thymine dimers are formed between non-adjacent thymines in UV-irradiated poly(dA-dT):poly(dA-dT). These are responsible for the incomplete recovery of DNA polymerase activity following heating due to their heat stability. Analyses of the irradiated and hydrolyzed substrate also demonstrated formation of minor yields of photoproducts formed by covalent linkage of adjacent thymines and adenines by UV-irradiation. Therefore, the thymine hydrates formed in UV-irradiated DNA partially inhibit polymerase activity during DNA synthesis and thus could be potentially lethal if unrepaired.     

The role of phosphoric acid residues in the formation of antigenic determinants of the structural components of DNA

The inhibitory activity of thymidine, thymidine triphosphate and thymidyl oligonucleotides was studied in thymidine-antithymidine antibodies reaction. Thymidine was shown to have the greatest inhibitory effect, with thymidine triphosphate and thymidyl oligonucleotide inhibitory activity less expressed and reducing with the increase in oligonucleotide length. The effect of thymidine, thymidine triphosphate and thymidyl oligonucleotides on the interaction of antisera and SLE patients' sera with denatured DNA was studied. It was shown that thymidine triphosphate and particularly thymidyl oligonucleotides are characterized by greater inhibitory capacity, as compared to thymidine. It was found that only thymine dimers bound by phosphate groups can inhibit the interaction of UV-irradiated DNA with antiserum specific for UV-modified DNA. The data obtained suggest that the charge determined by phosphoric acid residues plays an essential role in the interaction of antibodies induced to charged structural DNA components.     

Detection by electron microscopy of photo-induced denaturation in lambda DNA.

We have used an electron microscope to study localized denatured regions in ultraviolet-irradiated DNA. DNA from bacteriophage lambda was UV-irradiated and then prepared for electron microscopy after fixing in buffered (pH 9.5) formaldehyde solutions at 25 degrees C. The denatured regions observed corresponded to those described by Inman and Schnös (1) who used alkaline denaturation to preferentially destroy thymine-adenine base pairing. In UV-irradiated DNA, pairs of neighboring thymine residues are converted into photodimers; hence, loss of hydrogen bonding most likely occurs in thymine-rich regions and denaturation results. Conceivably, photo-induced denaturation may under some circumstances represent a more convenient method than alkaline denaturation for mapping thymine-rich regions in DNA.     

Demonstration of pyrimidine dimer-DNA glycosylase activity in vivo: bacteriophage T4-infected Escherichia coli as a model system.

An approach to the detection of pyrimidine dimer-DNA glycosylase activity in living cells is presented. Mutants of Escherichia coli defective in uvr functions required for incision of UV-irradiated DNA were infected with phage T4 denV+ or denV- (defective in the T4 pyrimidine dimer-DNA glycosylase activity). In the former case the denV gene product catalyzed the incision of UV-irradiated host DNA, facilitating the subsequent excision of thymine-containing pyrimidine dimers. Isolation of these dimers from the acid-soluble fraction of infected cells was achieved by a multistep thin-layer chromatographic system. Exposure of the dimers to irradiation that monomerizes pyrimidine dimers (direct photoreversal) resulted in the stoichiometric formation of free thymine. Thus, in vivo incision of UV-irradiated DNA dependent on a pyrimidine dimer-DNA glycosylase can be demonstrated.     

Enhancement of the uvrA gene dosage reduces pyrimidine dimer excision in UV-irradiated Escherichia coli

E. coli possesses an efficient repair mechanism able to remove pyrimidine dimers from UV-irradiated DNA, which is catalyzed by UvrABC endonuclease. In E. coli B/r Hcr⁺ cells transformed with a multicopy plasmid harboring a gene coding for UvrA, the excision capacity was greatly reduced. The course of thymine dimer excision was investigated using the enzymatic as well as the radiochromatographic method and the results are discussed in term of nonspecific interaction between the excess of UvrA protein and undamaged DNA duplex.     

Photomonomerization of pyrimidine dimers by indoles and proteins.

Model systems for the study of photoreactivation have been developed that utilize a variety of indole derivatives. These systems can split uracil cis-syn cyclobutadipyrimidine, either free or in RNA, when irradiated at wave-lengths absorbed only by the indole moiety. The ability of indole compounds to split dimers is closely related to their electronic properties. Those of high electron-donor capacity such as indole, 3-methylindole, indole-3-acetic acid, 5-hydroxytryptophan and tryptophan are good photosensitizers, with efficacy in that order. Indoles with electron-withdrawing substituents such as indole-3-carboxylic acid, indole-3-aldehyde and oxindole are inactive in the monomerization reaction. These findings support the proposed mechanism that the photosensitized monomerization occurs as a result of electron transfer from the excited indole molecules to the pyrimidine bases.Proteins containing fully exposed tryptophan residues (chicken egg white lysozyme and bovine diisopropylphosphoryltrypsin) also cause the splitting of the ¹⁴C-labeled dimers under the same conditions. In the case of lysozyme the quantum yield of monomerization is similar to that of free tryptophan. Much of the monomerization ability of lysozyme was lost after the solvent-available tryptophan had been oxidized by treatment with N-bromosuccinimide. Bovine pancreatic ribonuclease A, a protein devoid of tryptophan, failed to exhibit photosensitized monomerization of uracil dimers. The biological implication of these reactions involving a protein with an exposed tryptophan residue is discussed.Although indoles are able to split the dimers in RNA, they fail to photo-reactivate u.v.-damaged TMV-RNA. Indole-3-acetic acid, 3-methylindole and 5-hydroxytryptophan rapidly inactivate viral RNA when irradiated at 313 nm, possibly because of side reactions.     

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.     

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.     

Reactivity of antibodies to guanosine modified by the carcinogen N-acetoxy-N-2-acetylaminofluorene.

N-(guanosin-8-yl) acetylaminofluorene (Guo-AAF) was prepared by the reaction of N-acetoxy-N-2-acetylaminofluorene (AAAF) and guanosine. Antibodies to Guo-AAF were elicited in rabbits by immunization with bovine serum albumin-Guo-AAF conjugate. The antibodies were purified by affinity chromatography on a Sepharose-Guo-AAF column. The reactivity of these antibodies towards several ligands was studied by radioimmunoassay. The antibodies have the same affinity for double stranded DNA-AAF and single stranded DNA-AAF. Thus the geometry of the regions of DNA substituted by AAF residues is the same in native and denatured DNA. The affinity of the antibodies is smaller for DNA-AAF than for Guo-AAF. This can be due in part to the stacking of AAF residues with the adjacent bases as shown by the study of the interactions between the antibodies and AAF-oligonucleotides. The circular dichroism spectra of AAF-oligonucleotides bound to the antibodies are reported.     

Mechanisms of targeted frameshift mutations: Insertions arising during error-prone or SOS synthesis of DNA containing cis-syn cyclobutane thymine dimers

It is still unclear how frameshift mutations arise at cyclobutane pyrimidine dimers. The polymerase model is commonly used to explain the mechanisms of various mutations. An alternative polymerase-tautomer model was developed for UV-induced mutagenesis. A mechanism was proposed for targeted insertions caused by cis-syn cyclobutane thymine dimers. Targeted insertions are frameshift mutations due to addition of one or more nucleotides in a DNA sequence opposite to a lesion capable of stopping DNA synthesis. Among other factors, cyclobutane pyrimidine dimers can cause targeted insertions. UV irradiation can change the tautomeric form of DNA bases. Five rare tautomeric forms are possible for thymine, and they are stable when the thymine is a component of a cyclobutane dimer. A structural analysis showed that none of the canonical nucleotides can be added opposite to a specific rare thymine tautomer so that hydrogen bonds form between the two bases. A single nucleotide gap is consequently left in the corresponding site of the nascent strand when a specialized or modified DNA polymerase drives SOS or error-prone DNA synthesis on a template containing cis-syn cyclobutane thymine dimers with a base occurring in the rare tautomeric form. If the DNA composition is homogenous within the region, the end of the growing DNA strand may slip to form a complementary pair with the nucleotide adjacent to the dimer according to the Streisinger model, thus producing a loop. A targeted insertion is thereby generated to make the daughter strand longer. Targeted insertions were for the first time assumed to result from the cis-syn cyclobutane thymine dimers wherein one or both of the bases occur in the specific tautomeric form that does not allow the addition and hydrogen bonding of any canonical nucleotide in the opposite position. A model was developed to explain how targeted insertions of one or more nucleotides are caused by cis-syn cyclobutane thymine dimers. Thus, the polymerase-tautomer model can explain the nature and formation of targeted frameshift mutations in addition to hot and cold spots or targeted or untargeted nucleotide substitutions.     

Substrate overlap and functional competition between human nucleotide excision repair and Escherichia coli photolyase and (a)BC excision nuclease

Human cell free extract prepared by the method of Manley et al. (1980) carries out repair synthesis on UV-irradiated DNA. Removal of pyrimidine dimers by photoreactivation with DNA photolyase reduces repair synthesis by about 50%. With excess enzyme in the reaction mixture photolyase reduced the repair signal by the same amount even in the absence of photoreactivating light, presumably by binding to pyrimidine dimers and interfering with the binding of human damage recognition protein. Similarly, the UvrB subunit of Escherichia coli (A)BC excinuclease when loaded onto UV-irradiated or psoralen-adducted DNA inhibited repair synthesis by cell-free extract by 75-80%. The opposite was true also as HeLa cell free extract specifically inhibited the photorepair of a thymine dimer by DNA photolyase and its removal by (A)BC excinuclease. Cell-free extracts from xeroderma pigmentosum (XP) complementation groups A and C were equally effective in blocking the E. coli repair proteins, while extracts from complementation groups D and E were ineffective in blocking the E. coli enzyme. These results suggest that XP-D and XP-E cells are defective in the damage recognition subunit(s) of human excision nuclease.     

Location of the T4 gene 32 protein-binding site on polyoma virus DNA

Three easily denatured regions can be demonstrated in polyoma virus DNA. T4 gene 32 protein which binds to single stranded DNA, but not to duplex DNA, will specifically bind to any of these sites when viral DNA is in its superhelical configuration. These sites were mapped relative to a unique E. coli R_I endonuclease cleavage site by electron microscopy.     

The termini of bacteriophage T7 DNA

The termini of Escherichia coli phage T7 DNA have been labeled with ³²P by the polynucleotide kinase reaction. The DNA was fragmented, denatured, and annealed to denatured T7 DNA embedded in agar; elution was measured as a function of temperature. The terminal fragments were eluted from the gel at temperatures well below that of the bulk of the DNA, suggesting that these regions have a very high adenine-plus-thymine content. However, when DNA doubly labeled throughout at random by growth of the phage in [³H]thymidine and ³²PO₄, was denatured, annealed to the gel, and eluted as a function of temperature, the material eluting from the gel in this low-temperature range was about 50% adenine and thymine. Hence the melting behavior of the terminal fragments is not a result of a high adenine plus thymine content. By electrophoretic analysis of exonucleolytic digests of the T7 DNA it was shown that no unusual bases were present. It is suggested that the low thermal stability of the annealed terminal fragments is a consequence of the high guanine·cytosine regions being unavailable for hybridization, possibly because they are involved in intra-strand hydrogen bonding.     

Photoreactivation in airborne Mycobacterium parafortuitum

Photoreactivation was observed in airborne Mycobacterium parafortuitum exposed concurrently to UV radiation (254 nm) and visible light. Photoreactivation rates of airborne cells increased with increasing relative humidity (RH) and decreased with increasing UV dose. Under a constant UV dose with visible light absent, the UV inactivation rate of airborne M. parafortuitum cells decreased by a factor of 4 as RH increased from 40 to 95%; however, under identical conditions with visible light present, the UV inactivation rate of airborne cells decreased only by a factor of 2. When irradiated in the absence of visible light, cellular cyclobutane thymine dimer content of UV-irradiated airborne M. parafortuitum and Serratia marcescens increased in response to RH increases. Results suggest that, unlike in waterborne bacteria, cyclobutane thymine dimers are not the most significant form of UV-induced DNA damage incurred by airborne bacteria and that the distribution of DNA photoproducts incorporated into UV-irradiated airborne cells is a function of RH.     

Mechanisms and implications of the age-associated decrease in DNA repair capacity.

Skin cancer incidence is clearly linked to UV irradiation and increases exponentially with age. We studied the rate of removal of thymine dimers and (6-4) photoproducts in UV-irradiated human dermal fibroblasts derived from donors of different ages. There was a significant decrease with aging in the repair rates of both thymine dimers and (6-4) photoproducts (P<0.001). In addition, there was an age-associated decrease in the protein levels of ERCC3, PCNA, RPA, XPA, and p53 that participate in nucleotide excision repair. Moreover, the mRNA levels of XPA, ERCC3, and PCNA were significantly reduced with aging, suggesting that these decreases are often regulated at the mRNA level. Furthermore, with age induction of p53 after UV irradiation was significantly reduced. Taken together, our data suggest that the age-associated decrease in the repair of UV-induced DNA damage results at least in part from decreased levels of proteins that participate in the repair process.     

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.