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.     

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.     

A nonuniform distribution of excision repair synthesis in nucleosome core DNA

We have investigated the distribution in nucleosome core DNA of nucleotides incorporated by excision repair synthesis occurring immediately after UV irradiation in human cells. We show that the differences previously observed for whole nuclei between the DNase I digestion profiles of repaired DNA (following its refolding into a nucleosome structure) and bulk DNA are obtained for isolated nucleosome core particles. Analysis of the differences obtained indicates that they could reflect a significant difference in the level of repair-incorporated nucleotides at different sites within the core DNA region. To test this possibility directly, we have used exonuclease III digestion of very homogeneous sized core particle DNA to "map" the distribution of repair synthesis in these regions. Our results indicate that in a significant fraction of the nucleosomes the 5' and 3' ends of the core DNA are markedly enhanced in repair-incorporated nucleotides relative to the central region of the core particle. A best fit analysis indicates that a good approximation of the data is obtained for a distribution where the core DNA is uniformly labeled from the 5' end to position 62 and from position 114 to the 3' end, with the 52-base central region being devoid of repair-incorporated nucleotides. This distribution accounts for all of the quantitative differences observed previously between repaired DNA and bulk DNA following the rapid phase of nucleosome rearrangement when it is assumed that linker DNA and the core DNA ends are repaired with equal efficiency and the nucleosome structure of newly repaired DNA is identical with that of bulk chromatin. Furthermore, the 52-base central region that is devoid of repair synthesis contains the lowest frequency cutting sites for DNase I in vitro, as well as the only "internal" locations where two (rather than one) histones interact with a 10-base segment of each DNA strand.     

DNase I site mapping and micrococcal nuclease digestion of pachytene chromatin reveal novel structural features

A comparison of the DNase I digestion products of the 32P-5'-end-labeled pachytene nucleosome core particles (containing histones H2A, TH2A, X2, H2B, TH2B, H3, and H4) and liver nucleosome core particles (containing somatic histones H2A, H2B, H3, and H4) revealed that the cleavage sites that are 30, 40, and 110 nucleotides away from the 5'-end are significantly more accessible in the pachytene core particles than in the liver core particles. These cleavage sites correspond to the region wherein H2B interacts with the nucleosome core DNA. These results, therefore, suggest that the histone-DNA interaction at these sites in the pachytene core particles is weaker, possibly because of the presence of the histone variant TH2B interacting at similar topological positions in the nucleosome core as that of its somatic counterpart H2B. Such a loosened structure may also be maintained even in the native pachytene chromatin since micrococcal nuclease digestion of pachytene nuclei resulted in a higher ratio of subnucleosomes (SN4 + SN7) to mononucleosomes than that observed in liver chromatin.     

Enhanced DNA repair synthesis in hyperacetylated nucleosomes

We have investigated the level of "early" DNA repair synthesis in nucleosome subpopulations, varying in histone acetylation, from normal human fibroblasts treated with sodium butyrate. We find that repair synthesis occurring during the first 30 min after UV irradiation is significantly enhanced in hyperacetylated mononucleosomes. Nucleosomes with an average of 2.3 acetyl residues/H4 molecule contained approximately 1.8-fold more repair synthesis than nucleosomes with an average of 1.5 or 1.0 acetyl residues/H4 molecule. Fractionation of highly acetylated nucleosomes by two-dimensional gel electrophoresis yielded an additional 2.0-fold enrichment of repair synthesis in nucleosomes containing 2.7 acetyl residues/H4 molecule as compared to nucleosomes containing 1.9 acetyl residues/H4 molecule. This enhanced repair synthesis is associated primarily with nucleosome core regions and does not appear to result from increased UV damage in hyperacetylated chromatin. In addition, the distribution of repair synthesis within nucleosome core DNA from hyperacetylated chromatin is nonrandom, showing a bias toward the 5' end which is similar to that obtained for bulk (unfractionated) chromatin. These results provide strong evidence that enhanced repair occurs within nucleosome cores of hyperacetylated chromatin in butyrate-treated human cells. Finally, pulse-chase experiments demonstrate that the association of enhanced repair synthesis with hyperacetylated nucleosomes is transient, lasting only about 12 h after UV damage.     

DNA base excision repair of uracil residues in reconstituted nucleosome core particles

The human base excision repair machinery must locate and repair DNA base damage present in chromatin, of which the nucleosome core particle is the basic repeating unit. Here, we have utilized fragments of the Lytechinus variegatus 5S rRNA gene containing site-specific U:A base pairs to investigate the base excision repair pathway in reconstituted nucleosome core particles in vitro. The human uracil-DNA glycosylases, UNG2 and SMUG1, were able to remove uracil from nucleosomes. Efficiency of uracil excision from nucleosomes was reduced 3- to 9-fold when compared with naked DNA, and was essentially uniform along the length of the DNA substrate irrespective of rotational position on the core particle. Furthermore, we demonstrate that the excision repair pathway of an abasic site can be reconstituted on core particles using the known repair enzymes, AP-endonuclease 1, DNA polymerase beta and DNA ligase III. Thus, base excision repair can proceed in nucleosome core particles in vitro, but the repair efficiency is limited by the reduced activity of the uracil-DNA glycosylases and DNA polymerase beta on nucleosome cores.     

Stability of nucleosome placement in newly repaired regions of DNA

Rearrangements of chromatin structure during excision repair of UV-damaged DNA appear to involve unfolding of nucleosomal DNA while repair is taking place, followed by refolding of this DNA into a native nucleosome structure. Recently, we found that repair patches are not distributed uniformly along the DNA in nucleosome core particles immediately following their refolding into nucleosomes (Lan, S. Y., and Smerdon, M. J. (1985) Biochemistry, 24,7771). Therefore, the distribution of repair patches in nucleosome core DNA was used to monitor the stability of nucleosome placement in these regions. Our results indicate that in nondividing human cells undergoing excision repair there is a slow change in the positioning of nucleosomes in newly repaired regions of chromatin, resulting in the eventual randomization of repair patches in nucleosome core DNA. Furthermore, the nonrandom placement of nucleosomes observed just after the refolding event is not re-established during DNA replication. Possible mechanisms for this change in nucleosome placement along the DNA are discussed.     

UV Damage in DNA Promotes Nucleosome Unwrapping

The association of DNA with histones in chromatin impedes DNA repair enzymes from accessing DNA lesions. Nucleosomes exist in a dynamic equilibrium in which portions of the DNA molecule spontaneously unwrap, transiently exposing buried DNA sites. Thus, nucleosome dynamics in certain regions of chromatin may provide the exposure time and space needed for efficient repair of buried DNA lesions. We have used FRET and restriction enzyme accessibility to study nucleosome dynamics following DNA damage by UV radiation. We find that FRET efficiency is reduced in a dose-dependent manner, showing that the presence of UV photoproducts enhances spontaneous unwrapping of DNA from histones. Furthermore, this UV-induced shift in unwrapping dynamics is associated with increased restriction enzyme accessibility of histone-bound DNA after UV treatment. Surprisingly, the increased unwrapping dynamics is even observed in nucleosome core particles containing a single UV lesion at a specific site. These results highlight the potential for increased “intrinsic exposure” of nucleosome-associated DNA lesions in chromatin to repair proteins.     

大鼠老化过程大脑皮层及小脑神经元染色质构象分析

 本文在前文~[2]的基础上进一步以MCN和DNaseⅠ为探针研究大鼠脑神经元终末分化后不同生理时期染色质构象,结果表明:MCN酶解DNA产物PAGE显示脑老化过程大脑皮层及小脑神经元染色质核小体单体DNA分别保持在176bp和215bp水平,核小体连接DNA长度存在组织差异,但不受老化影响;<2>DNaseⅠ酶解DNA产物PAGE显示各年龄组大脑皮层及小脑神经元染色质DNA存在10bp间隔重复结构和相同的泳动区带分布特征,提示脑老化中染色质具有稳定的B型双螺旋结构和一致的螺线管卷曲形式。染色质DNaseⅠ降解率随年龄增加而降低,提示老化导致活性染色质区域减少,老化过程脑神经元染色质构象改变成为其转录功能减退的结构基础。     

Nucleotide excision repair from site-specifically platinum-modified nucleosomes

Nucleotide excision repair is a major cellular defense mechanism against the toxic effects of the anticancer drug cisplatin and other platinum-based chemotherapeutic agents. In this study, mononucleosomes were prepared containing either a site-specific cis-diammineplatinum(II)-DNA intrastrand d(GpG) or a d(GpTpG) cross-link. The ability of the histone core to modulate the excision of these defined platinum adducts was investigated as a model for exploring the cellular response to platinum-DNA adducts in chromatin. Comparison of the extent of repair by mammalian cell extracts of free and nucleosomal DNA containing the same platinum-DNA adduct reveals that the nucleosome significantly inhibits nucleotide excision repair. With the GTG-Pt DNA substrate, the nucleosome inhibits excision to about 10% of the level observed with free DNA, whereas with the less efficient GG-Pt DNA substrate the nucleosome inhibited excision to about 30% of the level observed with free DNA. The effects of post-translational modification of histones on excision of platinum damage from nucleosomes were investigated by comparing native and recombinant nucleosomes containing the same intrastrand d(GpTpG) cross-link. Excision from native nucleosomal DNA is approximately 2-fold higher than the level observed with recombinant material. This result reveals that post-translational modification of histones can modulate nucleotide excision repair from damaged chromatin. The in vitro system established in this study will facilitate the investigation of platinum-DNA damage by DNA repair processes and help elucidate the role of specific post-translational modification in NER of platinum-DNA adducts at the physiologically relevant nucleosome level.     

Chromatin remodeling facilitates DNA incision in UV-damaged nucleosomes

The DNA repair machinery must locate and repair DNA damage all over the genome. As nucleosomes inhibit DNA repair in vitro, it has been suggested that chromatin remodeling might be required for efficient repair in vivo. To investigate a possible contribution of nucleosome dynamics and chromatin remodeling to the repair of UV-photoproducts in nucleosomes, we examined the effect of a chromatin remodeling complex on the repair of UV-lesions by Micrococcus luteus UV endonuclease (ML-UV endo) and T4-endonuclease V (T4-endoV) in reconstituted mononucleosomes positioned at one end of a 175-bp long DNA fragment. Repair by ML-UV endo and T4-endoV was inefficient in mononucleosomes compared with naked DNA. However, the human nucleosome remodeling complex, hSWI/SNF, promoted more homogeneous repair by ML-UV endo and T4-endo V in reconstituted nucleosomes. This result suggests that recognition of DNA damage could be facilitated by a fluid state of the chromatin resulting from chromatin remodeling activities.     

DNA repair within nucleosome cores of UV-irradiated human cells

We have compared the distributions of repair synthesis and pyrimidine dimers (PD) in nucleosome core DNA during the early (fast) repair phase and the late (slow) repair phase of UV-irradiated human fibroblasts. As shown previously [Lan, S. Y., & Smerdon, M. J. (1985) Biochemistry 24, 7771-7783], repair synthesis is nonuniform in nucleosome core particles during the fast repair phase, and the distribution curve can be approximated by a model where repair synthesis occurs preferentially in the 5' and 3' end regions. In this report, we show that, during the slow repair phase, [3H]dThd-labeled repair patches are much more uniformly distributed in core DNA, although they appear to be preferentially located in sequences degraded slowly by exonuclease III. This change in distribution cannot be explained by an increase in patch size during slow repair, since the size of these patches actually decreases to about half the size measured during the fast repair phase. Furthermore, PD mapping within core DNA at the single-nucleotide level demonstrated that, at least within the 30-130-base region from the 5' end, there is little (or no) selective removal of PD during the fast repair phase. However, the nonuniform distribution of repair synthesis obtained during fast repair throughout most of the core DNA region (approximately 40-146 bases) is accounted for by the nonuniform distribution of PD in core DNA. The near-uniform distribution of repair synthesis observed during slow repair may result from more extensive nucleosome rearrangement and/or nucleosome modification during this phase.     

Nucleosome rearrangement in human cells following short patch repair of DNA damaged by bleomycin.

We have examined the structure of newly repaired regions of chromatin in intact and permeabilized human cells following exposure to bleomycin (BLM). The average repair patch size (in permeabilized cells) was six to nine bases, following doses of 1-25 micrograms/mL BLM, and greater than 80% of the total repair synthesis was resistant to aphidicolin. In both intact and permeabilized cells, nascent repair patches were initially very sensitive to staphylococcal nuclease, analogous to repair induced by "long patch" agents, and are nearly absent from isolated nucleosome core DNA. Unlike long patch repair, however, the loss of nuclease sensitivity during subsequent chase periods was very slow in intact cells, or in permeabilized cells treated with a low dose of BLM (1 microgram/mL), and was abolished by treatment with hydroxyurea (HU) or aphidicolin (APC). The rate of repair patch ligation did not correlate with this slow rate of chromatin rearrangement since greater than 95% of the patches were ligated within 6 h after incorporation (even in the presence of HU or APC). In permeabilized cells, repair patches induced by either 5 or 25 micrograms/mL BLM, where significant levels of strand breaks occur in compact regions of chromatin, lost the enhanced nuclease sensitivity at a rate similar to that observed following long patch repair. This rapid rate of rearrangement was not affected by APC. These results indicate that short patch repair in linker regions of nucleosomes, and/or "open" regions of chromatin, involves much less nucleosome rearrangement than long patch repair or short patch repair in condensed chromatin domains.     

Chromatin rearrangements during nucleotide excision repair

The removal of DNA damage from the eukaryotic genome requires DNA repair enzymes to operate within the complex environment of chromatin. We review the evidence for chromatin rearrangements during nucleotide excision repair and discuss the extent and possible molecular mechanisms of these rearrangements, focusing on events at the nucleosome level of chromatin structure.     

Nucleosome structure and repair of N-methylpurines in the GAL1-10 genes of Saccharomyces cerevisiae

Nucleosome structure and repair of N-methylpurines were analyzed at nucleotide resolution in the divergent GAL1-10 genes of intact yeast cells, encompassing their common upstream-activating sequence. In glucose cultures where genes are repressed, nucleosomes with fixed positions exist in regions adjacent to the upstream-activating sequence, and the variability of nucleosome positioning sharply increases with increasing distance from this sequence. Galactose induction causes nucleosome disruption throughout the region analyzed, with those nucleosomes close to the upstream-activating sequence being most striking. In glucose cultures, a strong correlation between N-methylpurine repair and nucleosome positioning was seen in nucleosomes with fixed positions, where slow and fast repair occurred in nucleosome core and linker DNA, respectively. Galactose induction enhanced N-methylpurine repair in both strands of nucleosome core DNA, being most dramatic in the clearly disrupted, fixed nucleosomes. Furthermore, N-methylpurines are repaired primarily by the Mag1-initiated base excision repair pathway, and nucleotide excision repair contributes little to repair of these lesions. Finally, N-methylpurine repair is significantly affected by nearest-neighbor nucleotides, where fast and slow repair occurred in sites between pyrimidines and purines, respectively. These results indicate that nucleosome positioning and DNA sequence significantly modulate Mag1-initiated base excision repair in intact yeast cells.     

Histone H3 K56 acetylation, chromatin assembly, and the DNA damage checkpoint

The role of chromatin and its modulation during DNA repair has become increasingly understood in recent years. A number of histone modifications that contribute towards the cellular response to DNA damage have been identified, including the acetylation of histone H3 at lysine 56. H3 K56 acetylation occurs normally during S phase, but persists in the presence of DNA damage. In the absence of this modification, cellular survival following DNA damage is impaired. Two recent reports provide additional insights into how H3 K56 acetylation functions in DNA damage responses. In particular, this modification appears to be important for both normal replication-coupled nucleosome assembly as well as nucleosome assembly at sites of DNA damage following repair.     

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.     

The SWI/SNF chromatin-remodeling factor stimulates repair by human excision nuclease in the mononucleosome core particle

To investigate the role of chromatin remodeling in nucleotide excision repair, we prepared mononucleosomes with a 200-bp duplex containing an acetylaminofluorene-guanine (AAF-G) adduct at a single site. DNase I footprinting revealed a well-phased nucleosome structure with the AAF-G adduct near the center of twofold symmetry of the nucleosome core. This mononucleosome substrate was used to examine the effect of the SWI/SNF remodeling complex on the activity of human excision nuclease reconstituted from six purified excision repair factors. We found that the three repair factors implicated in damage recognition, RPA, XPA, and XPC, stimulate the remodeling activity of SWI/SNF, which in turn stimulates the removal of the AAF-G adduct from the nucleosome core by the excision nuclease. This is the first demonstration of the stimulation of nucleotide excision repair of a lesion in the nucleosome core by a chromatin-remodeling factor and contrasts with the ACF remodeling factor, which stimulates the removal of lesions from internucleosomal linker regions but not from the nucleosome core.