Ultraviolet-induced mutations in Cockayne syndrome cells are primarily caused by cyclobutane dimer photoproducts while repair of other photoproducts is normal.

We compared the contribution to mutagenesis in Cockayne syndrome (CS) cells of the major class of UV photoproducts, the cyclobutane pyrimidine dimer, to that of other DNA photoproducts by using the mutagenesis shuttle vector pZ189. Lymphoblastoid cell lines from the DNA repair-deficient disorders CS and xeroderma pigmentosum (XP) and a normal line were transfected with UV-treated pZ189. Cyclobutane dimers were selectively removed before transfection by photoreactivation (PR), leaving nondimer photoproducts intact. After UV exposure and replication in CS and XP cells, plasmid survival was abnormally reduced and mutation frequency was abnormally elevated. After PR, plasmid survival increased and mutation frequency in CS cells decreased to normal levels but remained abnormal in XP cells. Sequence analysis of > 200 mutant plasmids showed that with CS cells a major mutational hot spot was caused by unrepaired cyclobutane dimers. These data indicate that with both CS and XP cyclobutane dimers are major photoproducts generating reduced plasmid survival and increased mutation frequency. However, unlike XP, CS cells are proficient in repair of nondimer photoproducts. Since XP but not CS patients have a high frequency of UV-induced skin cancers, our data suggest that prevention of UV-induce skin cancers is associated with proficient repair of nondimer photoproducts.     

Nicking-closing enzyme assembles nucleosome-like structures in vitro.

The four core histones (H2A, H2B, H3, and H4) and DNA were assembled into nucleosome-like particles at physiological ionic strengths either by an extract of chromatin rich in nicking-closing activity or by the purified nicking-closing enzyme itself. When histone-DNA complexes were assembled in vitro from relaxed circular DNA, nearly physiological numbers of superhelical turns were induced in the DNA molecule. Electron microscopy of the complexes assembled by the chromatin extract revealed a beaded structure and a reduction of the contour length compared to free DNA. Micrococcal nuclease digestion of the histone-DNA complexes yielded 145-base-pair DNA fragments typical of nucleosome core particles and shorter subnucleosomal DNA fragments of discrete length.     

Crosslinked histone octamer as a model of the nucleosome core.

When histones in chromatin core particles were crosslinked with dimethylsuberimidate, the resulting particles had properties closely similar to those of native core particles. A crosslinked octameric histone complex was isolated from these particles under nondenaturing conditions. Upon reaction with DNA, this octameric protein folded the DNA into a structure closely resembling that of native core particles as verified by various techniques; protein denaturants were necessary for reassociation. The histone octamer is useful as a model of the nucleosome protein core and for studying histone-DNA interactions that occur in nucleosomes.     

Fast, long-range, reversible conformational fluctuations in nucleosomes revealed by single-pair fluorescence resonance energy transfer

The nucleosome core particle, the basic repeated structure in chromatin fibers, consists of an octamer of eight core histone molecules, organized as dimers (H2A/H2B) and tetramers [(H3/H4)2] around which DNA wraps tightly in almost two left-handed turns. The nucleosome has to undergo certain conformational changes to allow processes that need access to the DNA template to occur. By single-pair fluorescence resonance energy transfer, we demonstrate fast, long-range, reversible conformational fluctuations in nucleosomes between two states: fully folded (closed), with the DNA wrapped around the histone core, or open, with the DNA significantly unraveled from the histone octamer. The brief excursions into an extended open state may create windows of opportunity for protein factors involved in DNA transactions to bind to or translocate along the DNA.     

Extracts of Drosophila embryos mediate chromatin assembly in vitro.

Extracts of Drosophila embryos can mediate the assembly of a chromatinlike structure from histones and DNA under physiological conditions. The histone-DNA complex formed in vitro contains micrococcal nuclease-sensitive sites spaced at 200-base pair intervals. More extensive digestion of the complex by micrococcal nuclease generates 11S particles which cosediment with nucleosome core particles isolated from native chromatin. These particles contain 140-base pair DNA fragments which upon further cleavage with micrococcal nuclease give rise to a pattern of discretely sized DNA fragments characteristic of nucleosome core particles. We have assayed the chromatin assembly process both qualitatively by measuring the induction of supertwists into a relaxed circular DNA (a process requiring a nicking-closing enzyme) and quantitatively by measuring the formation of micrococcal nuclease-resistant DNA fragments from radioactively labeled linear DNA. The amount of chromatin formed depends primarily on the amount of histones, whereas the rate of assembly depends on the amount of extract protein added. The factors in the extract that mediate chromatin assembly appear to interact first with the DNA because preincubation of the DNA with the extract markedly increases the extent of assembly.     

Removal of UV light-induced pyrimidine-pyrimidone(6-4) products from Escherichia coli DNA requires the uvrA, uvrB, and urvC gene products.

Ultraviolet light induces the formation of cyclobutane pyrimidine dimers and pyrimidine- pyrimidone (6-4) photoproducts in cellular DNA. In Escherichia coli, the uvrA, uvrB, and uvrC genes are necessary for excision of cyclobutane dimers. To determine whether the uvrABC gene products are required for (6-4) product removal from DNA, a sensitive HPLC assay was developed that allows the separation and quantitation of both types of photoproducts. Both the T T cyclobutane dimer and the T-C(6-4) product were completely removed from the DNA after 2 hr of repair in a wild-type strain. Both products were also removed in the wild-type strain in the presence of chloramphenicol, an inhibitor of protein synthesis. No decrease in the amount of either T T cyclobutane dimer or of T-C(6-4) products was observed in strains that were deficient in any one of the three uvr gene products under similar conditions. We conclude the uvrABC enzyme complex is required for excision of (6-4) photoproducts from E. coli DNA.     

Pyrimidine dimers in DNA initiate systemic immunosuppression in UV-irradiated mice.

Exposing the skin of mice to UV radiation interferes with the induction of delayed and contact hypersensitivity immune responses initiated at nonirradiated sites. The identity of the molecular target in the skin for these immunosuppressive effects of UV radiation remains controversial. To test the hypothesis that DNA is the target for UV-induced systemic immunosuppression, we exposed C3H mice to UV radiation and then used liposomes to deliver a dimer-specific excision repair enzyme into the epidermis in situ. The application of T4 endonuclease V encapsulated in liposomes to UV-irradiated mouse skin decreased the number of cyclobutane pyrimidine dimers in the epidermis and prevented suppression of both delayed and contact hypersensitivity responses. Moreover, the formation of suppressor lymphoid cells was inhibited. Control, heat-inactivated endonuclease encapsulated in liposomes had no effect. These studies demonstrate that DNA is the major target of UV radiation in the generation of systemic immunosuppression and suggest that the primary molecular event mediating these types of immunosuppression by UV radiation is the formation of pyrimidine dimers. Furthermore, they illustrate that the delivery of lesion-specific DNA repair enzymes to living skin after UV irradiation is an effective tool for restoring immune function and suggest that this approach may be broadly applicable to preventing other alterations caused by DNA damage.     

Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells.

The role of UV radiation-induced photoproducts in initiating base substitution mutations in human cells was examined by measuring photoproduct frequency distributions and mutations in a supF tRNA gene on a shuttle vector plasmid transfected into DNA repair-deficient cells (xeroderma pigmentosum, complementation group A) and into normal cells. Frequencies of cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts varied by as much as 80-fold at different dipyrimidine sites within the gene. All transition mutations occurred at dipyrimidine sites, predominantly at cytosine, with a 17-fold variation in mutation frequency between different sites. Removal of greater than 99% of the cyclobutane dimers by in vitro photoreactivation before transfection reduced the mutation frequency while preserving the mutation distribution, indicating that (i) cytosine-containing cyclobutane dimers were the major mutagenic lesions at these sites and (ii) cytosine-containing non-cyclobutane dimer photoproducts were also mutagenic lesions. However, at individual dipyrimidine sites neither the frequency of cyclobutane dimers nor the frequency of pyrimidine-pyrimidone (6-4) photoproducts correlated with the mutation frequency, even in the absence of excision repair. Mutation hot spots occurred at sites with low or high frequency of photoproduct formation and mutation cold spots occurred at sites with many photoproducts. These results suggest that although photoproducts are required for UV mutagenesis, the prominence of most mutation hot spots and cold spots is primarily determined by DNA structural features rather than by the frequency of DNA photoproducts.     

Perturbation of maintenance and de novo DNA methylation in vitro by UVB (280-340 nm)-induced pyrimidine photodimers.

The effect of pyrimidine photodimers on transmethylation reactions catalyzed by a highly purified rat liver DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) that exhibits maintenance and de novo methylation activities was studied in vitro, using the viral substrates M13 mp9 replicative form (RF) DNA and the hemimethylated analog formed from primed synthesis of phage DNA in the presence of 2'-deoxy-5-methylcytidine 5'-triphosphate. These DNAs were irradiated with UVB (280-340 nm) at 900-3600 J/m2 in the presence of the triplet-state sensitizers acetone or 3-dimethylaminopropiophenone. Under these conditions of irradiation, which approximate solar UV, pyrimidine cyclobutane photodimers were introduced without producing any evidence of single-strand breaks or alkali-sensitive sites [i.e., no (6-4)pyrimidine-pyrimidone photoproducts]. This was confirmed by gel analysis, a T4 UV endonuclease nicking assay specific for cyclobutane-type dimers, and HPLC analysis of the photoproducts. The methylation of irradiated templates by DNA methyltransferase was inhibited in an approximately linear fashion as a function of increasing UVB dose. This inhibition was correlated with the number of lethal photoproducts detected by the simultaneous measurement of the surviving fraction of infectious phage DNA. For approximately the same number of pyrimidine cyclobutane photoproducts introduced, de novo methylation activity was approximately 2-fold more sensitive than the maintenance mode of methylation. The ability of these putatively carcinogenic, pyrimidine photoproducts to inhibit DNA methylation suggests a common mechanism of action with several chemical carcinogens that are known to modify bases.     

Rearrangement of the histone H2A C-terminal domain in the nucleosome.

Using zero-length covalent protein-DNA crosslinking, we have mapped the histone-DNA contacts in nucleosome core particles from which the C- and N-terminal domains of histone H2A were selectively trimmed by trypsin or clostripain. We found that the flexible trypsin-sensitive C-terminal domain of histone H2A contacts the dyad axis, whereas its globular domain contacts the end of DNA in the nucleosome core particle. The appearance of the histone H2A contact at the dyad axis occurs only in the absence of linker DNA and does not depend on the absence of linker histones. Our results show the ability of the histone H2A C-terminal domain to rearrange. This rearrangement might play a biological role in nucleosome disassembly and reassembly and the retention of the H2A-H2B dimer (or the whole octamer) during the passing of polymerases through the nucleosome.     

A possible explanation for the nuclease limit digestion pattern of chromatin.

The general pattern of DNA fragments in the limit digest of nuclease-treated chromatin could arise from a single, unique nuclease-susceptible site per nucleosome. If DNA binds to the histone core of the nucleosome along a circularly re-entrant path, the location of the DNA entrance and exit can occur at any of a number of distinct sites. This very specific type of heterogeneity together with the natural 10-fold periodicity of DNA B can account for the observed digestion pattern. Such a general picture of the nucleosome structure could also easily explain how nucleosomes might move along the DNA. This type of structure should be easy to distinguish experimentally form more conventional explanations of the origin of the limit digest pattern of chromatin.     

Distribution of UV light-induced damage in a defined sequence of human DNA: detection of alkaline-sensitive lesions at pyrimidine nucleoside-cytidine sequences.

The distribution of UV light-induced damage to the highly reiterated alpha sequence of human DNA was investigated. The results show that the distribution of UV light-induced cyclobutane dimers within a defined sequence is similar whether the DNA is exposed to UV light as part of the chromosome of intact cells or as naked DNA. However, the cellular environment shields the nuclear DNA, resulting in about 50% decrease in apparent dose. A new type of UV photodamage was detected. Treatment of UV light-irradiated DNA with hot alkali results in strand breaks at positions of cytidine located 3' to pyrimidine nucleosides. The chemical nature and biological significance of the pyrimidine nucleoside-cytidine lesion is discussed.     

Mechanism of SOS mutagenesis of UV-irradiated DNA: mostly error-free processing of deaminated cytosine.

We measured the kinetics of growth and mutagenesis of UV-irradiated DNA of phages S13 and lambda that were undergoing SOS repair; the kinetics strongly suggest that most of SOS mutagenesis arises from the deamination of cytosine in cyclobutane pyrimidine dimers, producing C----T transitions. This occurs because the SOS mechanism bypasses T--T dimers promptly, while bypass of cytosine-containing dimers is delayed long enough for deamination to occur. The mutations are thus primarily the product of a faithful mechanism of lesion bypass by a DNA polymerase and are not, as had been generally thought, the product of an error-prone mechanism. All of these observations are explained by the A-rule, which is that adenine nucleotides are inserted noninstructionally opposite DNA lesions.     

The C-C (6-4) UV photoproduct is mutagenic in Escherichia coli.

Mutation induced by ultraviolet light is predominantly targeted by UV photoproducts. Two primary candidates for the premutagenic lesion are the cyclobutane pyrimidine dimer and the less frequent (by a factor of 10) pyrimidine-pyrimidone (6-4) photoproduct. Methylation of the 3'-cytosine in the sequence 5' CCAGG 3' reduces the yield of (6-4) lesions, but not of cyclobutane dimers, at these sites. By taking advantage of mutants deficient in cytosine methylation, we show here that at the three sites in the lacI gene of Escherichia coli having this sequence, the specific increase in the formation of the (6-4) photoproducts is accompanied by a concomitant increase in mutation. At each site, a G X C to A X T transition results in an amber mutation. In the unmethylated state, these sites become among the most frequent nonsense mutations recovered. We conclude that the (6-4) photoproduct constitutes a major premutagenic lesion in E. coli.     

Escherichia coli DNA photolyase stimulates uvrABC excision nuclease in vitro.

Pyrimidine dimers are the major photoproducts produced in cellular DNA upon UV irradiation. In Escherichia coli there are dark and photorepair mechanisms that eliminate the dimers from DNA and prevent their lethal and mutagenic effects. To determine whether these repair mechanisms act cooperatively or competitively in repairing DNA, we investigated the effects upon one another of DNA photolyase, which mediates photorepair, and uvrABC excision nuclease, an enzyme complex of the uvrABC gene products, which catalyzes nucleotide excision repair. We found that photolyase stimulates the removal of pyrimidine dimers but not other DNA adducts by uvrABC excision nuclease. The two subunits of uvrABC excision nuclease, the uvrA and uvrB proteins which together bind to the dimer region of DNA, had no effect on the activity of photolyase. T4 endonuclease V, which like photolyase is specific for pyrimidine dimers, was inhibited by photolyase, suggesting that these two proteins recognize the same or similar chemical structures in UV-irradiated DNA that are different from those recognized by uvrABC excision nuclease.     

Thymine-containing dimers as well as spore photoproducts are found in ultraviolet-irradiated Bacillus subtilis spores that lack small acid-soluble proteins.

Dormant spores of a Bacillus subtilis mutant that lacks two major small, acid-soluble spore proteins are very sensitive to UV irradiation, which in spores generates about half the amount of thymine-containing dimers formed by comparable irradiation of vegetative cells. Irradiation of mutant spores also produces spore photoproducts, but again only about one-half the amount formed in comparably irradiated wild-type spores. These findings suggest that the high UV sensitivity of the mutant spores is due to the production of pyrimidine dimers, which are not found in UV-irradiated wild-type spores, and that the high level of small, acid-soluble proteins found in wild-type spores is directly involved in spore UV resistance by facilitating a conformational change in spore DNA, preventing pyrimidine dimer formation.     

Mechanism of ultraviolet-induced mutagenesis: Extent and fidelity of in vitro DNA synthesis on irradiated templates

The effect of UV irradiation on the extent and fidelity of DNA synthesis in vitro was studied by using homopolymers and primed single-stranded varphiX174 phage DNA as substrates. Unfractionated and fractionated cell-free extracts from Escherichia coli pol(+) and polA1 mutants as well as purified DNA polymerase I were used as sources of enzymatic activity. (DNA polymerases, as used here, refer to deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7.) The extent of inhibition of DNA synthesis on UV-irradiated varphiX174 DNA suggested that pyrimidine dimers act as an absolute block for chain elongation by DNA polymerases I and III. Experiments with an irradiated poly(dC) template failed to detect incorporation of noncomplementary bases due to pyrimidine dimers. A large increase in the turnover of nucleoside triphosphates to free monophosphates during synthesis by DNA polymerase I on irradiated varphiX174 DNA has been observed. We propose that this nucleotide turnover is due to idling by DNA polymerase (i.e., incorporation and subsequent excision of nucleotides opposite UV photolesions, by the 3'-->5' "proofreading" exonuclease) thus preventing replication past pyrimidine dimers and the potentially mutagenic event that should result. In support of this hypothesis, DNA synthesis by DNA polymerase from avian myeloblastosis virus and by mammalian DNA polymerase alpha, both of which are devoid of any exonuclease activity, was found to be only partially inhibited, but not blocked, by UV irradiation of the template and accompanied by an increased incorporation of noncomplementary nucleotides. It is suggested that UV mutagenesis in bacteria requires an induced modification of the cellular DNA replication machinery, possibly an inhibition of the 3'-->5' exonuclease activity associated with DNA polymerases.     

Expression of the denV gene of bacteriophage T4 cloned in Escherichia coli

The denV gene of bacteriophage T4 has been cloned into Escherichia coli K-12 by inserting appropriate fragments of cytosine-containing T4 DNA into the Sal I site of the plasmid pBR322. The denV gene codes for an enzyme that initiates the excision repair of pyrimidine dimers produced in DNA by UV. In uvrA recA mutants, deficient in an early step in excision repair, the cloned DNA results in enhanced UV resistance that is more pronounced in stationary- than in exponential-phase cultures. The expression of the cloned DNA also results in the enhanced survival of UV-irradiated phage lambda or of a denV mutant of phage T4 and in removal of dimers from the DNA of UV-irradiated cells.     

Altered nucleosome structure containing DNA sequences complementary to 19S and 26S ribosomal RNA in Physarum polycephalum.

The localization of DNA sequences coding for ribosomal RNA was studied by hybridization of purified ribosomal RNA to DNA from chromatin fragments prepared by limited digestion of Physarum nuclei with staphylococcal nuclease. The 32P-labeled 19S and 26S RNA hybridized to DNA from nucleosome monomers, dimers, trimers, and higher oligomers, separated by sucrose gradient centrifugation, although the level of hybridization to DNA from nucleosome fractions was less than the level of hybridization to undigested nuclear DNA. The distribution of 19S and 26S rDNA sequences in the nucleosome fractions differed from the distribution of bulk DNA in that the rDNA sequences were recovered primarily in two fractions containing monomer-sized DNA lengths (140-160 base pairs). The percentage of DNA hybridizing to 19S plus 26S RNA was greater in peak A, the more slowly sedimenting monomer peak, than in any other chromatin fraction at all stages of digestion. Peak A and monomer particles differed in protein content and distribution. The presence of ribosomal cistrons in an altered nucleosome configuration may be related to changes in functional states of rDNA chromatin.