Rad23 is required for transcription-coupled repair and efficient overrall repair in Saccharomyces cerevisiae.

The repair of UV-induced photoproducts (cyclobutane pyrimidine dimers) in a well-characterized minichromosome, genomic DNA, and a transcribed genomic gene (RPB2) of a rad23delta mutant of Saccharomyces care was examined. Isogenic wild-type cells show a strong bias for the repair of the transcribed strands in both the plasmid and genomic genes and efficient overall repair of both DNAs (>80% of the dimers were removed in 6 h). However, the rad23delta mutant shows (i) no strand bias for repair in these genes and decreased repair of both strands, (ii) partial repair of genomic DNA (approximately 45% in 6 h), and (iii) very poor repair of the plasmid overall approximately 15% in 6 h). These features, coupled with the decreased UV survival of rad23delta cells, indicate that Rad23 is required for both transcription-coupled repair and efficient overall repair in S. cerevisiae.

Nucleotide excision repair in a constitutive and inducible gene of a yeast minichromosome in intact cells.

Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in a yeast minichromosome, having a galactose-inducible GAL1:URA3 fusion gene, a constitutively expressed HIS3 gene and varied regions of chromatin structure. Transcription of GAL1:URA3 increased >150-fold, while HIS3 expression decreased <2-fold when cells were switched from glucose to galactose medium. Following galactose induction, four nucleosomes were displaced or rearranged in the GAL3-GAL10 region. However, no change in nucleosome arrangement was observed in other regions of the minichromosome following induction, indicating that only a few plasmid molecules actively transcribe at any one time. Repair at 269 cis-syn CPD sites revealed moderate preferential repair of the transcribed strand of GAL1:URA3 in galactose, consistent with transcription-coupled repair in a fraction of these genes. Many sites upstream of the transcription start site in the transcribed strand were also repaired faster upon induction. There is remarkable repair heterogeneity in the HIS3 gene and preferential repair is seen only in a short sequence immediately downstream of the transcription start site. Finally, a mild correlation of repair heterogeneity with nucleosome positions was observed in the transcribed strand of the inactive GAL1:URA3 gene and this correlation was abolished upon galactose induction.

Modulation of DNA damage and DNA repair in chromatin.

DNA is packaged in the highly compact and dynamic structure of chromatin in eukaryotic cells. It is generally accepted that DNA processing events in the nucleus, such as transcription, replication, recombination, and repair, are restricted by this packaging. For some processes (e.g., transcription), the chromatin fiber is "preset" in a more open structure to allow access of proteins to specific regions of DNA within this structural hierarchy. These regions contain modified nucleosomes that accommodate a less compact state of chromatin and allow access to specific regions of DNA. DNA repair proteins, however, must access DNA lesions in all structural domains of chromatin after sudden insult to the genome. Damaged DNA must be recognized, removed, and replaced by repair enzymes at all levels of chromatin packaging. Therefore, the modulation of DNA damage and its repair in chromatin is crucial to our understanding of the fate of potential mutagenic and carcinogenic lesions in DNA. In this review, we discuss the modulation of DNA damage and DNA repair by chromatin structure, and the modulation of chromatin structure by these events.

Bleomycin-induced DNA damage and repair in human cells permeabilized with lysophosphatidylcholine.

We have examined bleomycin-induced DNA damage and repair in confluent human fibroblasts that were reversibly permeabilized to small molecules (e.g., deoxynucleotide triphosphates and trypan blue) by a short exposure to 80 micrograms/ml lysophosphatidylcholine. We found that this treatment dramatically increases the dose effectiveness of bleomycin in inducing DNA strand breaks and DNA repair synthesis in these cells. For example, when intact cells (not treated with lysophosphatidylcholine) were incubated with 100 micrograms/ml bleomycin, only about 5% of the cell population was observed to have undergone measurable DNA repair synthesis (by autoradiography). On the other hand, when these cells were reversibly permeabilized with lysophosphatidylcholine before treatment, we observed significant repair synthesis in greater than 80% of the cells using a bleomycin dose of only 5 micrograms/ml. Furthermore, sufficient levels of single- and double-strand breaks were introduced into nucleosome linker DNA of permeabilized cells to yield a nucleosomal repeat pattern in alkaline and neutral agarose gels. However, no change in the amount of DNA less than 23 kilobases was observed on these gels when intact cells were incubated with bleomycin.

Photofootprint of nucleosome core DNA in intact chromatin having different structural states.

Recently, we reported that the distribution of ultraviolet light (u.v.) induced pyrimidine dimers in nucleosome core DNA has a striking 10.3(+/- 0.1) base periodicity and the regions of enhanced quantum yield map to positions where DNA strands are farthest from the core histone surface. Improvement of the mapping procedure has allowed us to analyze this distribution in more detail, and compare the distribution pattern for nucleosome cores from intact chromatin having different higher-order structures (from the 10 nm filament to the 30 nm fiber). At all levels of chromatin compaction, we observed the following. (1) The average periodicity in pyrimidine dimer yield is 10.3 bases. (2) The peak-to-peak spacing in this distribution is significantly different from 10.3 bases in the region covering three helix turns immediately 5' of the dyad axis. (3) There is a suppression of photoproduct formation in the region of the dyad axis, especially at position 84 from the 5' end. (4) The approximately 10 base ensembles have alternating peak intensities throughout core DNA. Furthermore, peak deconvolution analysis of the pyrimidine dimer pattern yielded a striking similarity in photoproduct yield for the different levels of chromatin compaction. Irradiation of isolated core DNA yields a much more random distribution of photoproducts, although a weak modulation pattern is observed (indicating that there is a non-random alignment of adjacent pyrimidines in our core DNA preparations). This pattern includes a depression in photoproduct yield near position 95, suggesting that the sequence in this region plays a role in nucleosome positioning. These results show that the u.v. photofootprint is a sensitive, diagnostic probe of core histone-DNA interactions in intact chromatin, and these interactions are not significantly altered by changes in the structural state of the chromatin fiber.

Limited nucleosome migration can completely randomize DNA repair patches in intact human cells.

Following irradiation of human cells with ultraviolet light, DNA repair patches are initially inserted near the 5' and 3' ends of nucleosome core DNA leaving a "gap" in repair synthesis (of approximately 50 bases) near the center of the core DNA. With time, however, these same repair patches become randomized, apparently by nucleosome migration. We have developed both an analytical expression and a computer algorithm (which simulates nucleosome migration along DNA) to determine the average distance nucleosomes must migrate to change the initial, non-uniform distribution of repair patches in nucleosomes to a random distribution. Both of these methods yielded the same result: nucleosomes must migrate an average of about 50 base-pairs in either direction to produce the randomization observed.

Improved method for measuring the ensemble average of strand breaks in genomic DNA.

The cis-syn cyclobutane pyrimidine dimer (CPD) is the major photoproduct induced in DNA by low wavelength ultraviolet radiation. An improved method was developed to detect CPD formation and removal in genomic DNA that avoids the problems encountered with the standard method of endonuclease detection of these photoproducts. Since CPD-specific endonucleases make single-strand cuts at CPD sites, quantification of the frequency of CPDs in DNA is usually done by denaturing gel electrophoresis. The standard method of ethidium bromide staining and gel photography requires more than 10 microg of DNA per gel lane, and correction of the photographic signal for the nonlinear film response. To simplify this procedure, a standard Southern blot protocol, coupled with phosphorimage analysis, was developed. This method uses random hybridization probes to detect genomic sequences with minimal sequence bias. Because of the vast linearity range of phosphorimage detection, scans of the signal profiles for the heterogeneous population of DNA fragments can be integrated directly to determine the number-average size of the population.

UV induced (6-4) photoproducts are distributed differently than cyclobutane dimers in nucleosomes.

We have compared the distributions of two stable UV photoproducts in nucleosome core DNA at the single-nucleotide level using a T4 polymerase-exonuclease mapping procedure. The distribution of pyrimidine-pyrimidone (6-4) dimers was uncovered by reversing the major UV photo-product, cis-syn cyclobutane pyrimidine dimer, with E. coli DNA photolyase and photoreactivating light. Whereas the distribution of total UV photoproducts in nucleosome core DNA forms a striking 10.3 base periodic pattern, the distribution of (6-4) dimers is much more random throughout the nucleosome core domain. Therefore, histone-DNA interactions in nucleosomes strongly modulate formation of the major class of UV-induced photoproducts, while having either a constant effect or no effect on (6-4) dimer formation.

Excision repair of UV damage in human fibroblasts reversibly permeabilized by lysolecithin.

We have examined nucleotide excision repair synthesis in confluent human diploid fibroblasts permeabilized with lysolecithin. Following a UV dose of 12 J/m2, maximal incorporation of [alpha 35S]dNTPs occurred at a lysolecithin concentration (approximately 80 micrograms/ml) where slightly more than 90% of the cells were initially permeable to trypan blue. However, autoradiography of cells, permeabilized at this lysolecithin concentration, demonstrated that only about 20% of the total cell population incorporated significant levels of 35S into DNA. This result presumably reflected the fact that approximately 20% of the total cell population remained permeable for much longer periods of time (up to 2 h) than the remaining cell population (less than 20 min). The incorporation of dNTPs by UV-irradiated, permeabilized cells appeared to be bona fide excision repair synthesis since: (1) Incorporation was completely absent in unirradiated, permeabilized cells and in irradiated, permeabilized repair-deficient cells. (2) Nucleotides incorporated in the presence of BrdUTP were associated with normal density DNA. (3) The apparent Km for all 4 dNTPs was 50-100 nM, in agreement with past reports on human fibroblasts irreversibly permeabilized by cell lysis. (4) DNA associated with the newly incorporated dNTPs underwent ligation and rearrangements in chromatin structure analogous to what is observed in intact human cells. Repair incorporation of dNTPs was rapid and linear during the first 2 h after UV irradiation and permeabilization. After this time, incorporation ceased or continued at a much slower rate. Cell viability experiments and autoradiography demonstrated that the cells permeabilized to [3H]dNTPs were capable of carrying out DNA replication and cell division. Thus, confluent human diploid fibroblasts can be reversibly permeabilized to labeled dNTPs by lysolecithin for the study of excision repair following physiologic doses of UV radiation. However, under these conditions, only a fraction of the cells remain permeable for an extended period of time.

Mitotic viability and metabolic competence in UV-irradiated yeast cells.

Colony formation is the classic method for measuring survival of yeast cells. This method measures mitotic viability and can underestimate the fraction of cells capable of carrying out other DNA processing events. Here, we report an alternative method, based on cell metabolism, to determine the fraction of surviving cells after ultraviolet (UV) irradiation. The reduction of 2,3,5-triphenyl tetrazolium chloride (or TTC) to formazan in mitochondria was compared with cell colony formation and DNA repair capacity in wt cells and two repair-deficient strains (rad1Delta and rad7Delta). Both TTC reduction and cell colony formation gave a linear response with different ratios of mitotically viable cells and heat-inactivated cells. However, monitoring the formation of formazan in non-dividing yeast cells that are partially (rad7Delta) or totally (wt) proficient at DNA repair is a more accurate measure of cell survival after UV irradiation. Before repair of UV photoproducts (cis-syn cyclobutane pyrimidine dimers or CPDs) is complete, these two assays give very different results, implying that many damaged cells are metabolically competent but cannot replicate. For example, only 25% of the rad7Delta cells are mitotically viable after a UV dose of 12 J/m(2)75% of these cells are metabolically competent and remove over 55% of the CPDs from their genomic DNA. Moreover, repair of CPDs in wt cells dramatically decreases after the first few hours of liquid holding (L.H.; incubation in water) and correlates with a substantial decrease in cell metabolism over the same time period. In contrast, cell colony formation may be the more accurate indicator of cell survival after UV irradiation of rad1Delta cells (i.e., cells with little DNA repair activity). These results indicate that the metabolic competence of UV-irradiated, non-dividing yeast cells is a much better indicator of cell survival than mitotic viability in partially (or totally) repair proficient yeast cultures.

Completion of excision repair in human cells. Relationship between ligation and nucleosome formation.

The completion of excision repair patches in human cells, following UV irradiation, was compared to the refolding of these regions into nucleosomes. Incomplete repair patches were detected by their enhanced sensitivity to exonuclease III. This enhanced sensitivity was due to the presence of gaps (or displaced parental strands) at the 3' end rather than unligated nicks, indicating that ligation occurs rapidly after repair synthesis is completed. Different rates of completion were achieved by treatment with the inhibitors hydroxyurea and sodium butyrate, as well as by using a (partially) ligase-deficient human cell strain. Hydroxyurea caused a marked decrease in both the rate of completion and the level of repair incorporation in all three cell types studied, while sodium butyrate yielded different effects in each cell type. In each case, however, a decrease in the rate of repair patch completion resulted in a concomitant decrease in the level of nucleosome formation. To determine the temporal relationship of these two events, the levels of repair-incorporated nucleotides in isolated 146-base pair nucleosome core DNA were compared on native and denaturing gels. The data indicate that little (or no) nucleosome formation occurred in the nascent DNA regions prior to ligation regardless of the cell type or treatment used. Furthermore, comparison of the fraction of unligated repair patches and the fraction of repair patches in a nonnucleosomal state indicated that in the absence of inhibitors there was a significant time lag between ligation and nucleosome formation. This lag time, however, decreased when cells were treated with hydroxyurea. Thus, the formation of nucleosomes in newly repaired regions of DNA occurred after the ligation step in all cases and these two features of the excision repair process are not "tightly coupled" events.

Rearrangements of chromatin structure in newly repaired regions of deoxyribonucleic acid in human cells treated with sodium butyrate or hydroxyurea.

The rate and extent of redistribution of repair-incorporated nucleotides within chromatin during very early times (10-45 min) after ultraviolet irradiation were examined in normal human fibroblasts treated with 20 mM sodium butyrate, or 2-10 mM hydroxyurea, and compared to results for untreated cells. Under these conditions, DNA replicative synthesis is reduced to very low levels in each case. However, DNA repair synthesis is stimulated by sodium butyrate and partially inhibited by hydroxyurea. Furthermore, in the sodium butyrate treated cells, the core histones are maximally hyperacetylated. Using methods previously described by us, it was found that treatment with sodium butyrate had little or no effect on either the rate or the extent of redistribution of repair-incorporated nucleotides during this early time interval. On the other hand, there was a 1.7-2.5-fold decrease in the rate of redistribution of these nucleotides in cells treated with hydroxyurea; the extent of redistribution was unchanged in these cells. Since hydroxyurea has been shown to decrease the rate of completion of "repair patches" in mammalian cells, these results indicate that nucleosome rearrangement in newly repaired regions of DNA does not occur until after the final stages of the excision repair process are completed. Furthermore, hyperacetylation of the core histones in a large fraction of the total chromatin prior to DNA damage and repair synthesis does not appear to alter the rate or extent of nucleosome core formation in newly repaired regions of DNA.

UV-induced pyrimidine dimers and trimethylpsoralen cross-links do not alter chromatin folding in vitro.

We have examined the ability of intact and histone H1 depleted chromatin fibers to fold into higher ordered structures in vitro following DNA damage by two different agents: UV irradiation at 254 nm and trimethylpsoralen plus near-UV light. Both agents damage DNA specifically, yet cause different degrees of unwinding (and possibly bending) of the DNA helix. In addition, trimethylpsoralen forms interstrand DNA cross-links. The structural transitions of intact and histone H1 depleted chromatin fibers, induced by NaCl, were monitored by analytical ultracentrifugation, light scattering, and circular dichroism. Our results indicate that when chromatin fibers contain even large, nonphysiological amounts of DNA photodamage by either agent, the salt-induced folding of these fibers into higher ordered structures is unaffected. The compact 30-nm fiber must therefore be able to accommodate a large amount of DNA photodamage (greater than one UV-induced photoproduct or trimethylpsoralen interstrand cross-link per nucleosome) with little or no change in the overall size or compaction of this structure.

Rearrangement of nucleosome structure during excision repair in xeroderma pigmentosum (group A) human fibroblasts.

Rearrangements of chromatin structure during excision repair were examined in xeroderma pigmentosum (XP; complementation group A) human fibroblasts treated with the small-molecule alkylating agent methyl methanesulfonate (MMS). In agreement with past reports, we observed normal levels of repair synthesis in these cells during the first 12 h after exposure to 1.5 mM MMS, in contrast to the near zero incorporation of repair patches following exposure to 12 J/m2 u.v. light. Our results indicate that the relative nuclease sensitivity of newly repaired regions in MMS-treated nuclease sensitivity of newly repaired regions in MMS-treated XP (group A) cells is quantitatively similar to that of newly repaired regions in MMS-treated normal human fibroblasts. This enhanced sensitivity is accompanied by a marked under-representation of repair-incorporated nucleotides in isolated nucleosome core DNA. Pulse-chase experiments demonstrated that these regions rapidly undergo rearrangements in chromatin structure, and both the rate and extent of these rearrangements are similar (but not identical) to those observed in normal cells. This was also the case for the rate and extent of ligation of repair patches, as measured by the sensitivity of these regions to exonuclease III digestion. If the changes in nuclease sensitivity of newly repaired regions in DNA reflect an unfolding of nucleosome structure during excision repair, then these results indicate that the activity associated with this unfolding is present in XP (group A) cells.

Site-specific DNA repair at the nucleosome level in a yeast minichromosome.

The rate of excision repair of UV-induced pyrimidine dimers (PDs) was measured at specific sites in each strand of a yeast minichromosome containing an active gene (URA3), a replication origin (ARS1), and positioned nucleosomes. All six PD sites analyzed in the transcribed URA3 strand were repaired more rapidly (greater than 5-fold on average) than any of the nine PD sites analyzed in the nontranscribed strand. Efficient repair also occurred in both strands of a disrupted TRP1 gene (ten PD sites), containing four unstable nucleosomes, and in a nucleosome gap at the 5' end of URA3 (two PD sites). Conversely, slow repair occurred in both strands immediately downstream of the URA3 gene (12 of 14 PD sites). This region contains the ARS1 consensus sequence, a nucleosome gap, and two stable nucleosomes. Thus, modulation of DNA repair occurs in a simple yeast minichromosome and correlates with gene expression, nucleosome stability, and (possibly) control of replication.

Distribution within chromatin of deoxyribonucleic acid repair synthesis occurring at different times after ultraviolet radiation.

We have compared the initial distribution and subsequent redistribution within chromatin of nucleotides incorporated during the early ("rapid") phase and the late ("slow") phase of UV-induced DNA repair synthesis. As has been observed for the early repair phase, most or all of the nucleotides incorporated during the late repair phase are initially staphylococcal nuclease and DNase I "sensitive" (i.e., rapidly digested). This initial enhanced sensitivity is accompanied by both an underrepresentation of these nucleotides in the 145--165 base pari (core) DNA produced by staphylococcal nuclease digestion and an absence of these nucleotides in the approximately 20-base repeat pattern produced by DNase I digestion. Furthermore, nucleotides incorporated at late time after damage are involved in nucleosome rearrangements as reported previously for repair synthesis occurring at early times [Smerdon, M. J., & Lieberman, M. W. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4238--4241]. The kinetics of redistribution, however, appear to be more rapid than those observed for early times. Following redistribution the average nucleosome repeat length of DNA containing repair-incorporated nucleotides is the same as that of bulk DNA regardless of the time after damage that repair occurs; also, many of these nucleotides coelectrophorese with the approximately 10-base repeat fragments generated by DNase I. The results yield a new interpretation of our previous studies [Smerdeon, M. J., Tlsty, T. D., & Lieberman, M. W. (1978) Biochemistry 17, 2377--2386] on the distribution of nucleotides incorporated at long times after UV irradiation.

UV damage to DNA strongly influences its rotational setting on the histone surface of reconstituted nucleosomes.

The major photoproduct formed in DNA, following absorption of ultraviolet (UV) light, is the cis-syn cyclobutane pyrimidine dimer (CPD). Formation of CPDs in DNA packaged into chromatin prior to UV irradiation results in a striking preference for these photoproducts to be oriented away from the histone surface in nucleosomes (Gale, J. M., Nissen, K. A., and Smerdon, M. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6644-6648). In this report, we show that formation of nucleosomes onto UV-irradiated DNA results in a similar distribution of these photoproducts, indicating that preexisting CPDs in the DNA molecule can influence its rotational setting on the histone surface during nucleosome formation. This bias is less pronounced in the central three helical turns encompassing the dyad axis of nucleosomes, where the helix is overwound (10.7 bases/turn) relative to the "outer" portion of core DNA (10.0 bases/turn). Such a change in the rotational setting of DNA on the surface of newly formed nucleosomes could expose normally inaccessible DNA sequences to factors which control DNA processing.