Nucleotide excision repair of the 5 S ribosomal RNA gene assembled into a nucleosome

A-175-base pair fragment containing the Xenopus borealis somatic 5 S ribosomal RNA gene was used as a model system to determine the effect of nucleosome assembly on nucleotide excision repair (NER) of the major UV photoproduct (cyclobutane pyrimidine dimer (CPD)) in DNA. Xenopus oocyte nuclear extracts were used to carry out repair in vitro on reconstituted, positioned 5 S rDNA nucleosomes. Nucleosome structure strongly inhibits NER at many CPD sites in the 5 S rDNA fragment while having little effect at a few sites. The time course of CPD removal at 35 different sites indicates that >85% of the CPDs in the naked DNA fragment have t(12) values <2 h, whereas <26% of the t(12) values in nucleosomes are <2 h, and 15% are >8 h. Moreover, removal of histone tails from these mononucleosomes has little effect on the repair rates. Finally, nucleosome inhibition of repair shows no correlation with the rotational setting of a 14-nucleotide-long pyrimidine tract located 30 base pairs from the nucleosome dyad. These results suggest that inhibition of NER by mononucleosomes is not significantly influenced by the rotational orientation of CPDs on the histone surface, and histone tails play little (or no) role in this inhibition.     

Synthesis and nucleosome structure of DNA containing a UV photoproduct at a specific site.

A strategy was developed to assemble nucleosomes specifically damaged at only one site and one structural orientation. The most prevalent UV photoproduct, a cis-syn cyclobutane thymine dimer (cs CTD), was chemically synthesized and incorporated into a 30 base oligonucleotide harboring the glucocorticoid hormone response element. This oligonucleotide was assembled into a 165 base pair double stranded DNA molecule with nucleosome positioning elements on each side of the cs CTD-containing insert. Proton NMR verified that the synthetic photoproduct is the cis-syn stereoisomer of the CTD. Moreover, two different pyrimidine dimer-specific endonucleases cut approximately 90% of the dsDNA molecules. This cleavage is completely reversed by photoreactivation with E. coli UV photolyase, further demonstrating the correct stereochemistry of the photoproduct. Nucleosomes were reconstituted by histone octamer exchange from chicken erythocyte core particles, and contained a unique translational and rotational setting of the insert on the histone surface. Hydroxyl radical footprinting demonstrates that the minor groove at the cs CTD is positioned away from the histone surface about 5 bases from the nucleosome dyad. Competitive gel-shift analysis indicates there is a small increase in histone binding energy required for the damaged fragment (DeltaDeltaG approximately 0.15 kcal/mol), which does not prevent complete nucleosome loading under our conditions. Finally, folding of the synthetic DNA into nucleosomes dramatically inhibits cleavage at the cs CTD by T4 endonuclease V and photoreversal by UV photolyase. Thus, specifically damaged nucleosomes can be experimentally designed for in vitro DNA repair studies.     

Activity of FEN1 endonuclease on nucleosome substrates is dependent upon DNA sequence but not flap orientation

We demonstrated previously that human FEN1 endonuclease, an enzyme involved in excising single-stranded DNA flaps that arise during Okazaki fragment processing and base excision repair, cleaves model flap substrates assembled into nucleosomes. Here we explore the effect of flap orientation with respect to the surface of the histone octamer on nucleosome structure and FEN1 activity in vitro. We find that orienting the flap substrate toward the histone octamer does not significantly alter the rotational orientation of two different nucleosome positioning sequences on the surface of the histone octamer but does cause minor perturbation of nucleosome structure. Surprisingly, flaps oriented toward the nucleosome surface are accessible to FEN1 cleavage in nucleosomes containing the Xenopus 5S positioning sequence. In contrast, neither flaps oriented toward nor away from the nucleosome surface are cleaved by the enzyme in nucleosomes containing the high-affinity 601 nucleosome positioning sequence. The data are consistent with a model in which sequence-dependent motility of DNA on the nucleosome is a major determinant of FEN1 activity. The implications of these findings for the activity of FEN1 in vivo are discussed.     

Nucleosome sliding induced by the xMi-2 complex does not occur exclusively via a simple twist-diffusion mechanism

ATP-dependent chromatin remodeling complexes can induce the translocation (sliding) of nucleosomes in cis along DNA, but the mechanism by which sliding occurs is not well defined. We previously presented evidence that sliding induced by the human SWI/SNF complex does not occur solely via a proposed "twist-diffusion" mechanism whereby the DNA rotates about its helical axis without displacement from the surface of the nucleosome (Aoyagi, S., and Hayes, J. J. (2002) Mol. Cell. Biol. 22, 7484-7490). Here we examined whether the Xenopus Mi-2 nucleosome remodeling complex induces nucleosome sliding via a twist-diffusion mechanism with nucleosomes assembled onto DNA templates containing branched DNA structures expected to sterically hinder rotation of the DNA helix on the nucleosome surface. We find that the branched DNA-containing nucleosomes undergo xMi-2-catalyzed sliding at a rate and extent identical to that of nucleosomes assembled on native DNA fragments. These results indicate that both the hSWI/SNF and xMi-2 complexes induce nucleosome sliding via a mechanism(s) other than simple twist diffusion and are consistent with models in which the DNA largely maintains its rotational orientation with respect to the histone surface.     

Ultraviolet damage and nucleosome folding of the 5S ribosomal RNA gene

The Xenopus borealis somatic 5S ribosomal RNA gene was used as a model system to determine the mutual effects of nucleosome folding and formation of ultraviolet (UV) photoproducts (primarily cis-syn cyclobutane pyrimidine dimers, or CPDs) in chromatin. We analyzed the preferred rotational and translational settings of 5S rDNA on the histone octamer surface after induction of up to 0.8 CPD/nucleosome core (2.5 kJ/m(2) UV dose). DNase I and hydroxyl radical footprints indicate that UV damage at these levels does not affect the average rotational setting of the 5S rDNA molecules. Moreover, a combination of nuclease trimming and restriction enzyme digestion indicates the preferred translational positions of the histone octamer are not affected by this level of UV damage. We also did not observe differences in the UV damage patterns of irradiated 5S rDNA before or after nucleosome formation, indicating there is little difference in the inhibition of nucleosome folding by specific CPD sites in the 5S rRNA gene. Conversely, nucleosome folding significantly restricts CPD formation at all sites in the three helical turns of the nontranscribed strand located in the dyad axis region of the nucleosome, where DNA is bound exclusively by the histone H3-H4 tetramer. Finally, modulation of the CPD distribution in a 14 nt long pyrimidine tract correlates with its rotational setting on the histone surface, when the strong sequence bias for CPD formation in this tract is minimized by normalization. These results help establish the mutual roles of histone binding and UV photoproducts on their formation in chromatin.     

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.     

Uracil DNA Glycosylase Activity on Nucleosomal DNA Depends on Rotational Orientation of Targets

The activity of uracil DNA glycosylases (UDGs), which recognize and excise uracil bases from DNA, has been well characterized on naked DNA substrates but less is known about activity in chromatin. We therefore prepared a set of model nucleosome substrates in which single thymidine residues were replaced with uracil at specific locations and a second set of nucleosomes in which uracils were randomly substituted for all thymidines. We found that UDG efficiently removes uracil from internal locations in the nucleosome where the DNA backbone is oriented away from the surface of the histone octamer, without significant disruption of histone-DNA interactions. However, uracils at sites oriented toward the histone octamer surface were excised at much slower rates, consistent with a mechanism requiring spontaneous DNA unwrapping from the nucleosome. In contrast to the nucleosome core, UDG activity on DNA outside the core DNA region was similar to that of naked DNA. Association of linker histone reduced activity of UDG at selected sites near where the globular domain of H1 is proposed to bind to the nucleosome as well as within the extra-core DNA. Our results indicate that some sites within the nucleosome core and the extra-core (linker) DNA regions represent hot spots for repair that could influence critical biological processes.     

Initiation and bidirectional propagation of chromatin assembly from a target site for nucleotide excision repair.

To restore full genomic integrity in a eukaryotic cell, DNA repair processes have to be coordinated with the resetting of nucleosomal organization. We have established a cell-free system using Drosophila embryo extracts to investigate the mechanism linking de novo nucleosome formation to nucleotide excision repair (NER). Closed-circular DNA containing a uniquely placed cisplatin-DNA adduct was used to follow chromatin assembly specifically from a site of NER. Nucleosome formation was initiated from a target site for NER. The assembly of nucleosomes propagated bidirectionally, creating a regular nucleosomal array extending beyond the initiation site. Furthermore, this chromatin assembly was still effective when the repair synthesis step in the NER process was inhibited.     

Distinct Features of the Histone Core Structure in Nucleosomes Containing the Histone H2A.B Variant

Nucleosomes containing a human histone variant, H2A.B, in an aqueous solution were analyzed by small-angle neutron scattering utilizing a contrast variation technique. Comparisons with the canonical H2A nucleosome structure revealed that the DNA termini of the H2A.B nucleosome are detached from the histone core surface, and flexibly expanded toward the solvent. In contrast, the histone tails are compacted in H2A.B nucleosomes compared to those in canonical H2A nucleosomes, suggesting that they bind to the surface of the histone core and/or DNA. Therefore, the histone tail dynamics may function to regulate the flexibility of the DNA termini in the nucleosomes.     

Distinct Features of the Histone Core Structure in Nucleosomes Containing the Histone H2A.B Variant

Nucleosomes containing a human histone variant, H2A.B, in an aqueous solution were analyzed by small-angle neutron scattering utilizing a contrast variation technique. Comparisons with the canonical H2A nucleosome structure revealed that the DNA termini of the H2A.B nucleosome are detached from the histone core surface, and flexibly expanded toward the solvent. In contrast, the histone tails are compacted in H2A.B nucleosomes compared to those in canonical H2A nucleosomes, suggesting that they bind to the surface of the histone core and/or DNA. Therefore, the histone tail dynamics may function to regulate the flexibility of the DNA termini in the nucleosomes.     

Nucleosomal Assembly in Vitro from Crypthecodinium cohnii Chromosomes in the Cell-free System

The typical eukaryote interphase nuclei were reconstructed in vitro following incubation of chromosomes of primitive eukaryote dinoflagellate Crypthecodinium cohnii E. in extracts of S phase eggs of Xenopus laevis L. Micrococcal nuclease digestion experiments indicated that the reconstructed nuclei contained typical nucleosomes. Since the chromosomes of C. cohnii do not contain histone and nucleosomes, these data suggest that nucleosome assembly is independent of specific DNA sequences, nuclear membranes and Lamin. Moreover, it has been demonstrated that Topoisomerase Ⅱ was partially involved in the process of nucleosome assembly. Taken together, these findings suggest that nuclear assembly is independent of formation of nucleosomes and that histone and non-histone may be the decisive factors in this process.     

Site-specific repair of cyclobutane pyrimidine dimers in a positioned nucleosome by photolyase and T4 endonuclease V in vitro.

Since genomic DNA is folded into nucleosomes, and DNA damage is generated all over the genome, a central question is how DNA repair enzymes access DNA lesions and how they cope with nucleosomes. To investigate this topic, we used a reconstituted nucleosome (HISAT nucleosome) as a substrate to generate DNA lesions by UV light (cyclobutane pyrimidine dimers, CPDs), and DNA photolyase and T4 endonuclease V (T4-endoV) as repair enzymes. The HISAT nucleosome is positioned precisely and contains a long polypyrimidine region which allows one to monitor formation and repair of CPDs over three helical turns. Repair by photolyase and T4-endoV was inefficient in nucleosomes compared with repair in naked DNA. However, both enzymes showed a pronounced site-specific modulation of repair on the nucleosome surface. Removal of the histone tails did not substantially enhance repair efficiency nor alter the site specificity of repair. Although photolyase and T4-endoV are different enzymes with different mechanisms, they exhibited a similar site specificity in nucleosomes. This implies that the nucleosome structure has a decisive role in DNA repair by exerting a strong constraint on damage accessibility. These findings may serve as a model for damage recognition and repair by more complex repair mechanisms in chromatin.     

Open, repair and close again: chromatin dynamics and the response to UV-induced DNA damage

Due to its link with human pathologies, including cancer, the mechanism of Nucleotide Excision Repair (NER) has been extensively studied. Most of the pathway and players have been defined using in vitro reconstitution experiments. However, in vivo, the NER machinery must deal with the presence of organized chromatin, which in some regions, such as heterochromatin, is highly condensed but still susceptible to DNA damage. A series of events involving different chromatin-remodeling factors and histone-modifying enzymes target chromatin regions that contain DNA lesions. CPDs change the structure of the nucleosome, allowing access to factors that can recognize the lesion. Next, DDB1-DDB2 protein complexes, which mono-ubiquitinate histones H2A, H3, and H4, recognize nucleosomes containing DNA lesions. The ubiquitinated nucleosome facilitates the recruitment of ATP-dependent chromatin-remodeling factors and the XPC-HR23B-Centrin 2 complex to the target region. Different ATP-dependent chromatin-remodeling factors, such as SWI/SNF and INO80, have been identified as having roles in the UV irradiation response prior to the action of the NER machinery. Subsequently, remodeling of the nucleosome allows enzymatic reactions by histone-modifying factors that may acetylate, methylate or demethylate specific histone residues. Intriguingly, some of these histone modifications are dependent on p53. These histone modifications and the remodeling of the nucleosome allow the entrance of TFIIH, XPC and other NER factors that remove the damaged strand; then, gap-filling DNA synthesis and ligation reactions are carried out after excision of the oligonucleotide with the lesion. Finally, after DNA repair, the initial chromatin structure has to be reestablished. Therefore, factors that modulate chromatin dynamics contribute to the NER mechanism, and they are significant in the future design of treatments for human pathologies related to genome instability and the appearance of drug-resistant tumors.     

Evidence That Nucleosomes Inhibit Mismatch Repair in Eukaryotic Cells

The influence of chromatin structure on DNA metabolic processes, including DNA replication and repair, has been a matter of intensive studies in recent years. Although the human mismatch repair (MMR) reaction has been reconstituted using purified proteins, the influence of chromatin structure on human MMR is unknown. This study examines the interaction between human MutSα and a mismatch located within a nucleosome or between two nucleosomes. The results show that, whereas MutSα specifically recognizes both types of nucleosomal heteroduplexes, the protein bound the mismatch within a nucleosome with much lower efficiency than a naked heteroduplex or a heterology free of histone proteins but between two nucleosomes. Additionally, MutSα displays reduced ATPase- and ADP-binding activity when interacting with nucleosomal heteroduplexes. Interestingly, nucleosomes block ATP-induced MutSα sliding along the DNA helix when the mismatch is in between two nucleosomes. These findings suggest that nucleosomes may inhibit MMR in eukaryotic cells. The implications of these findings for our understanding of eukaryotic MMR are discussed.     

Archaeal nucleosome positioning sequence from Methanothermus fervidus.

DNA in Methanothermus fervidus, a hyperthermophilic archaeon, is constrained into archaeal nucleosomes in vivo by the archaeal histones HMfA and HMfB. Here, we document the translational and rotational positioning of archaeal nucleosome assembly in vitro by a sequence from the 7S RNA encoding region of the M. fervidus genome. The minor groove of the DNA at the center of the DNA sequence, protected from micrococcal nuclease digestion by incorporation into a positioned archaeal nucleosome, faces away from the archaeal histone core.     

Sequence-specific positioning of core histones on an 860 base-pair DNA. Experiment and theory

Previous experiments have shown that the locations of the histone octamer on DNA molecules of 140 to 240 base-pairs (bp) are influenced strongly by the nucleotide sequence. Here we have studied the locations of the histone octamer on a relatively long DNA molecule of 860 bp, using two different nucleases, micrococcal and DNAase I. Data were obtained from both the protein--DNA complexes and from the naked DNA at single-bond resolution, and then were analyzed by densitometry to yield plots of differential cleavage, which show clearly the changes in cutting due to the addition of protein. Our results show that the placement of core histones on the 860 bp molecule is definitely non-random. The digestion data provide evidence for five nucleosome cores, the centers of which lie in defined locations. In all but one of these protein--DNA complexes, the DNA adopts a unique, highly preferred rotational setting with respect to the protein surface. Another protein--DNA complex is unusual in that it protects 200 bp from digestion, yet is cut in its very center as if it were split into two parts. The apparent average twist of the DNA within all of these protein--DNA complexes is 10.2(+/- 0.1) bp, as measured by the periodicity of DNAase I digestion. This value is in excellent agreement with the twist of 10.21(+/- 0.05) bp deduced from the periodicity of sequence content in chicken nucleosome core DNA. In addition, we observe a discontinuity in the periodic cutting by DNAase I of about -1 to -3 bonds in going from any nucleosome core to the next. The most plausible interpretation of this discontinuity is that it reflects the angle by which adjacent protein--DNA complexes are aligned. Thus, any nucleosome may be related to its neighbor by a left-handed rotation in space of -1/10.2 to -3/10.2 helix turns, or -35 degrees to -105 degrees. Repeated many times, this operation would build a long, left-handed helix of nucleosomes similar to that described by many workers for the packing of nucleosomes in chromatin. In order to look for any long-range influences on the positioning of the histone octamer in the 860 bp molecule (as would be expected if the nucleosomes have to fit into some higher-order structure), we have examined the locations of the histone octamer on five different isolated short fragments of the 860-mer, all of nucleosomal length.(ABSTRACT TRUNCATED AT 400 WORDS)