Repair-incorporated Nucleotides Research Articles

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.

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.

Nucleosome rearrangement in vitro. 2. Formation of nucleosomes in newly repaired regions of DNA.

We have reported previously that immediately following nucleotide excision repair in human cells the newly repaired DNA lacks a nucleosome conformation [Smerdon, M. J., & Lieberman, M. W. (1980) Biochemistry 19, 2992-3000]. In this study, we have examined the ability of these nascent DNA regions to acquire a nucleosome structure in vitro by incubating intact or H1-depleted nuclei in buffers containing different salt concentrations (0.025-0.625 M KCl) at 0 or 37 degrees C. Nucleosomes were detected in these regions by an increase in the level of repair-incorporated nucleotides associated with isolated nucleosome core particle DNA. Our results indicate that the nascent DNA is resistant to nucleosome formation during the low-salt transition where the limiting repeat length decreases from approximately 190 to 168 base pairs (bp) [Watkins, J. F., & Smerdon, M. J. (1985) Biochemistry (preceding paper in this issue)]. This result provides further evidence that the nascent DNA is indeed in a nonnucleosomal state. At higher salt concentrations (greater than 0.4 M), where the nucleosome repeat length decreases to a limiting value of approximately 146 bp, there was an increase in nucleosome formation in nascent DNA that correlated with the decrease in limiting repeat length. However, we did not observe a complete randomization of the repair-incorporated nucleotides. Indeed, even at the highest salt concentration used (0.625 M), we never observed more than 50% of the nascent DNA associated with the isolated core particles. This was the case even though a major portion of the nucleosomes had a limiting value repeat length following the high-salt incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple conformational states of repair patches in chromatin during DNA excision repair.

In mammalian cells, newly synthesized DNA repair patches are highly sensitive to digestion by staphylococcal nuclease (SN), but with time, they acquire approximately the same nuclease resistance as the DNA in bulk chromatin. We refer to the process which restores native SN sensitivity to repaired DNA as chromatin rearrangement. We find that during repair of ultraviolet damage in human fibroblasts, repair patch synthesis and ligation occur at approximately the same rate, with ligation delayed by about 4 min, but that chromatin rearrangement is only 75% as rapid. Thus, repair-incorporated nucleotides can exist in at least three distinct states: unligated/unrearranged, ligated/unrearranged, and ligated/rearranged. Inhibition of repair patch synthesis by aphidicolin or hydroxyurea results in inhibition of both patch ligation and chromatin rearrangement, confirming that repair patch completion and/or ligation are prerequisites for rearrangement. We also analyze the kinetics of SN digestion of repair-incorporated nucleotides at various extents of rearrangement and find the data to be consistent with the existence of two or more forms of unrearranged repair patch which have different sensitivities to digestion by SN. These data indicate that the chromatin rearrangement which restores native SN sensitivity to repaired DNA is a multistep process. The multiple forms of unrearranged chromatin with different SN sensitivities may include the unligated/unrearranged and ligated/unrearranged states. If so, the differences in SN sensitivity must arise from differences in chromatin structure, because SN does not differentiate between ligated and unligated repair patches in naked DNA.

Nuclease sensitivity of repair-incorporated nucleotides in chromatin and nucleosome rearrangement in human cells damaged by methyl methanesulfonate and methylnitrosourea.

We have examined both the initial nuclease sensitivity and subsequent nucleosome rearrangement of newly repaired regions of chromatin in human diploid fibroblasts treated with methyl methanesulfonate (MMS) and methylnitrosourea (MNU). We initially examined the effect of these two alkylating agents on DNA replicative synthesis. The results indicate that immediately following damage by MMS or MNU, at a concentration of 2 mM, the level of replicative synthesis is 20-25% of the level in untreated cells. In the MMS-treated cells, this suppression of replicative synthesis is short lived and by 15 h after damage the level of replicative synthesis is approximately 3-fold greater than that in untreated cells. This 'latent stimulation' of replicative synthesis was not observed in the cells treated with 2 mM MNU, although the level of replicative synthesis in these cells did approach the level of untreated cells at later times. When these contributions were corrected for, it was found that the nucleotides incorporated by repair synthesis are initially (i.e., immediately following repair synthesis) both staphylococcal nuclease and DNase I sensitive, and are underrepresented in isolated nucleosome core DNA. Using methods previously described by us, we show that the relative nuclease sensitivity of these regions is quantitatively similar to that of newly repaired DNA following damage by u.v. radiation. Furthermore, the relative nuclease sensitivity of newly repaired DNA is initially high regardless of the time after damage that repair occurs (at least for 13 h after damage). This feature is also similar to u.v. induced repair synthesis. Finally, pulse-chase experiments demonstrated that following repair synthesis induced by MMS or MNU rearrangements of chromatin structure take place, and both the rate and extent of these rearrangements are similar to that observed for cells treated with u.v. radiation or bulky chemical carcinogens. Thus, our results indicate that the excision repair induced by these two small alkylating agents is associated with the same overall chromatin structural features as the excision repair of DNA damage induced by u.v. radiation and 'u.v.-mimetic' chemicals.

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.

Rearrangement of mammalian chromatin structure following excision repair.

Repair of DNA damage in eukaryotic cells involves local disruption of chromatin structure such that repair-incorporated nucleotides are transiently highly sensitive to digestion by staphylococcal nuclease (SN)1–4. We have examined the dependence of this phenomenon on the initial distribution in chromatin of DNA damage by comparing the repair of pyrimidine dimers, which are distributed randomly in chromatin5, with the repair of angelicin photoadducts, which are formed primarily in linker regions of nucleosomes6,7. We find that the transient nuclease sensitivity of repair patches is independent of the initial distribution of DNA damage in chromatin. In addition, our observation that repair patches produced in response to a linker-specific damaging agent become distributed randomly in chromatin implies that nucleosome cores do not necessarily return to their original sites along a DNA strand after the DNA is repaired.

Alterations in chromatin structure during DNA excision repair.

Work from a number of laboratories recently has demonstrated that alterations in chromatin structure occur during excision repair in mammalian cells. It is now clear that when cells are damaged with a wide variety of chemical agents or ultraviolet radiation, almost all of the repair synthesis is initially sensitive to staphylococcal nuclease. With time, there is a redistribution of the counts incorporated during excision repair synthesis so that many of them become nuclease resistant and associated with nucleosome core length DNA. In our laboratory, we have demonstrated this phenomenon in human cells damaged with N-acetoxy-2-acetylaminofluorene, 7-bromomethylbenz[a]anthracene, and ultraviolet radiation. It is clear from the work of others that the phenomenon is not unique to human cells since African green monkey cells damaged with either ultraviolet radiation or angelicin also show an initial nuclease sensitivity of repair-incorporated nucleotides follow by rearrangement. Two models to explain these observations have been proposed; one suggests that there is an unfolding of nucleosomes during excision repair followed by a refolding, while the other suggests that sliding of core proteins with respect to DNA occurs during excision repair. These models, as well as recent data bearing on them, will be discussed.

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.

Distribution of repair-incorporated nucleotides and nucleosome rearrangement in the chromatin of normal and xeroderma pigmentosum human fibroblasts

Distribution of repair-incorporated nucleotides and nucleosome rearrangement in the chromatin of normal and xeroderma pigmentosum human fibroblasts

Distribution of DNA damage in chromatin and its relation to repair in human cells treated with 7-bromomethylbenz(a) anthracene.

We have examined the relationship between the distribution of DNA damage and repair in chromatin from confluent human fibroblasts treated with the carcinogen 7-bromomethylbenz (a) anthracene. Analysis of staphylococcal nuclease (SN)4 digestion kinetics and gel electrophoresis revealed that more total damage occurs in nucleosome core DNA (approximately 80-85% of chromatin DNA) than in SN sensitive DNA (APPROXIMATELY15-20%). Furthermore, over a 24 hr period, damage is removed at about the same rate from these two regions. In contrast, virtually all of the nucleotides incorporated during repair synthesis are initially SN sensitive even when measured at 12 hr after damage. With time many repair-incorporated nucleotides become SN resistant and coelectrophorese with nucleosome core DNA. To explain these data we propose a model whereby excision repair occurs in both linker and core DNA; however, in core DNA the repair process induces conformational changes resulting in temporarily increased SN sensitivity; subsequently, rearrangement occurs and results in the re-establishment of native or near-native nucleosome conformation and SN resistance.