Suppressed catalytic activity of base excision repair enzymes on rotationally positioned uracil in nucleosomes

The majority of DNA in eukaryotic cells exists in the highly condensed structural hierarchy of chromatin, which presents a challenge to DNA repair enzymes in that recognition, incision, and restoration of the original sequence at most sites must take place within these structural constraints. To test base excision repair (BER) activities on chromatin substrates, an in vitro system was developed that uses human uracil DNA glycosylase (UDG), apyrimidinic/apurinic endonuclease (APE), and DNA polymerase beta (pol beta) on homogeneously damaged, rotationally positioned DNA in nucleosomes. We find that UDG and APE carry out their combined catalytic activities with reduced efficiency on nucleosome substrates ( approximately 10% of that on naked DNA). Furthermore, these enzymes distinguish between two different rotational settings of the lesion on the histone surface, showing a 2- to 3-fold difference in activity between uracil facing "toward" and "away from" the histones. However, UDG and APE will digest such substrates to completion in a concentration-dependent manner. Conversely, the synthesis activity of pol beta is inhibited completely by nucleosome substrates and is independent of enzyme concentration. These results suggest that the first two steps of BER, UDG and APE, may occur "unassisted" in chromatin, whereas downstream factors in this pathway (i.e., pol beta) may require nucleosome remodeling for efficient DNA BER in at least some regions of chromatin in eukaryotic cells.     

Multistep pathway for replication-dependent nucleosome assembly.

We have used cell-free DNA replication to study the relationship between DNA replication and chromatin assembly. As others have reported, we find that DNA replication facilitates nucleosome assembly. We show here that replication-dependent nucleosome assembly occurs in at least two steps. The first step requires replicating DNA; the second step occurs after replication has been completed and is promoted by a nuclear extract. Consistent with this multistep model, we observe that the replicated simian virus 40 minichromosome is organized into a repeating array of DNA-protein particles that are structurally distinct from mature nucleosomes. These particles may be precursors in a pathway of nucleosome assembly since in the second, replication-independent step the nuclear extract converts this nascent chromatin into nucleosomes.     

Nonconservative segregation of parental nucleosomes during simian virus 40 chromosome replication in vitro.

Simian virus 40 chromosomes can be replicated in vitro with the same set of purified proteins required for the replication of naked DNA containing the viral origin. With these reconstituted systems, the fate of parental histones during replication was examined in vitro. The assembly of nucleosomes on replicating chromosomes was hardly affected by the presence of simultaneously replicating naked DNA competitor, suggesting that replication forks can traverse nucleosomes without the displacement of histones. Moreover, we demonstrate that the nascent nucleosomes were distributed almost equally between the leading and lagging strands. This distributive mode of nucleosome segregation favors the propagation of parental chromatin structures to both daughter cells, which can maintain cellular functions dictated by these structures during cell proliferation.     

Interplay between mismatch repair and chromatin assembly

Single strand nicks and gaps in DNA have been reported to increase the efficiency of nucleosome loading mediated by chromatin assembly factor 1 (CAF-1). However, on mismatch-containing substrates, these strand discontinuities are utilized by the mismatch repair (MMR) system as loading sites for exonuclease 1, at which degradation of the error-containing strand commences. Because packaging of DNA into chromatin might inhibit MMR, we were interested to learn whether chromatin assembly is differentially regulated on heteroduplex and homoduplex substrates. We now show that the presence of a mismatch in a nicked plasmid substrate delays nucleosome loading in human cell extracts. Our data also suggest that, once the mismatch is removed, repair of the single-stranded gap is accompanied by efficient nucleosome loading. We postulated that the balance between MMR and chromatin assembly might be governed by proliferating cell nuclear antigen (PCNA), the processivity factor of replicative DNA polymerases, which is loaded at DNA termini and which interacts with the MSH6 subunit of the mismatch recognition factor MutSα, as well as with CAF-1. We now show that this regulation might be more complex; MutSα and CAF-1 interact not only with PCNA, but also with each other. In vivo this interaction increases during S-phase and may be controlled by the phosphorylation status of the p150 subunit of CAF-1.     

Nucleosome assembly protein-1 is a linker histone chaperone in Xenopus eggs

In eukaryotic cells, genomic DNA is primarily packaged into nucleosomes through sequential ordered binding of the core and linker histone proteins. The acidic proteins termed histone chaperones are known to bind to core histones to neutralize their positive charges, thereby facilitating their proper deposition onto DNA to assemble the core of nucleosomes. For linker histones, however, little has been known about the regulatory mechanism for deposition of linker histones onto the linker DNA. Here we report that, in Xenopus eggs, the linker histone is associated with the Xenopus homologue of nucleosome assembly protein-1 (NAP-1), which is known to be a chaperone for the core histones H2A and H2B in Drosophila and mammalian cells [Ito, T., Bulger, M., Kobayashi, R. & Kadonaga, J. T. (1996) Mol. Cell Biol. 16, 3112-3124; Chang, L., Loranger, S. S., Mizzen, C., Ernst, S. G., Allis, C. D. & Annunziato, A. T. (1997) Biochemistry 36, 469-480]. We show that NAP-1 acts as the chaperone for the linker histone in both sperm chromatin remodeling into nucleosomes and linker histone binding to nucleosome core dimers. In the presence of NAP-1, the linker histone is properly deposited onto linker DNA at physiological ionic strength, without formation of nonspecific aggregates. These results strongly suggest that NAP-1 functions as a chaperone for the linker histone in Xenopus eggs.     

Centromere identity is specified by a single centromeric nucleosome in budding yeast

Chromosome segregation ensures that DNA is equally divided between daughter cells during each round of cell division. The centromere (CEN) is the specific locus on each chromosome that directs formation of the kinetochore, the multiprotein complex that interacts with the spindle microtubules to promote proper chromosomal alignment and segregation during mitosis. CENs are organized into a specialized chromatin structure due to the incorporation of an essential CEN-specific histone H3 variant (CenH3) in the centromeric nucleosomes of all eukaryotes. Consistent with its essential role at the CEN, the loss or up-regulation of CenH3 results in mitotic defects. Despite the requirement for CenH3 in CEN function, it is unclear how CenH3 nucleosomes structurally organize centromeric DNA to promote formation of the kinetochore. To address this issue, we developed a modified chromatin immunoprecipitation approach to analyze the number and position of CenH3 nucleosomes at the budding yeast CEN. Using this technique, we show that yeast CENs have a single CenH3 nucleosome positioned over the CEN-determining elements. Therefore, a single CenH3 nucleosome forms the minimal unit of centromeric chromatin necessary for kinetochore assembly and proper chromosome segregation.     

Nucleosomes, linker DNA, and linker histone form a unique structural motif that directs the higher-order folding and compaction of chromatin

The compaction level of arrays of nucleosomes may be understood in terms of the balance between the self-repulsion of DNA (principally linker DNA) and countering factors including the ionic strength and composition of the medium, the highly basic N termini of the core histones, and linker histones. However, the structural principles that come into play during the transition from a loose chain of nucleosomes to a compact 30-nm chromatin fiber have been difficult to establish, and the arrangement of nucleosomes and linker DNA in condensed chromatin fibers has never been fully resolved. Based on images of the solution conformation of native chromatin and fully defined chromatin arrays obtained by electron cryomicroscopy, we report a linker histone-dependent architectural motif beyond the level of the nucleosome core particle that takes the form of a stem-like organization of the entering and exiting linker DNA segments. DNA completes ≈1.7 turns on the histone octamer in the presence and absence of linker histone. When linker histone is present, the two linker DNA segments become juxtaposed ≈8 nm from the nucleosome center and remain apposed for 3–5 nm before diverging. We propose that this stem motif directs the arrangement of nucleosomes and linker DNA within the chromatin fiber, establishing a unique three-dimensional zigzag folding pattern that is conserved during compaction. Such an arrangement with peripherally arranged nucleosomes and internal linker DNA segments is fully consistent with observations in intact nuclei and also allows dramatic changes in compaction level to occur without a concomitant change in topology.     

Heterochromatin protein Sir3 induces contacts between the amino terminus of histone H4 and nucleosomal DNA

The regulated binding of effector proteins to the nucleosome plays a central role in the activation and silencing of eukaryotic genes. How this binding changes the properties of chromatin to mediate gene activation or silencing is not fully understood. Here we provide evidence that association of the budding yeast silent information regulator 3 (Sir3) silencing protein with the nucleosome induces a conformational change in the amino terminus of histone H4 that promotes interactions between the conserved H4 arginines 17 and 19 (R17 and R19) and nucleosomal DNA. Substitutions of H4R17 and R19 with alanine abolish silencing in vivo, but have little or no effect on binding of Sir3 to nucleosomes or histone H4 peptides in vitro. Furthermore, in both the previously reported crystal structure of the Sir3-bromo adjacent homology (BAH) domain bound to the Xenopus laevis nucleosome core particle and the crystal structure of the Sir3-BAH domain bound to the yeast nucleosome core particle described here, H4R17 and R19 make contacts with nucleosomal DNA rather than with Sir3. These results suggest that Sir3 binding generates a more stable nucleosome by clamping H4R17 and R19 to nucleosomal DNA, and raise the possibility that such induced changes in histone–DNA contacts play major roles in the regulation of chromatin structure.     

Tripartite organization of centromeric chromatin in budding yeast

The centromere is the genetic locus that organizes the proteinaceous kinetochore and is responsible for attachment of the chromosome to the spindle at mitosis and meiosis. In most eukaryotes, the centromere consists of highly repetitive DNA sequences that are occupied by nucleosomes containing the CenH3 histone variant, whereas in budding yeast, a ～120-bp centromere DNA element (CDE) that is sufficient for centromere function is occupied by a single right-handed histone variant CenH3 (Cse4) nucleosome. However, these in vivo observations are inconsistent with in vitro evidence for left-handed octameric CenH3 nucleosomes. To help resolve these inconsistencies, we characterized yeast centromeric chromatin at single base-pair resolution. Intact particles containing both Cse4 and H2A are precisely protected from micrococcal nuclease over the entire CDE of all 16 yeast centromeres in both solubilized chromatin and the insoluble kinetochore. Small DNA-binding proteins protect CDEI and CDEIII and delimit the centromeric nucleosome to the ～80-bp CDEII, only enough for a single DNA wrap. As expected for a tripartite organization of centromeric chromatin, loss of Cbf1 protein, which binds to CDEI, both reduces the size of the centromere-protected region and shifts its location toward CDEIII. Surprisingly, Cse4 overproduction caused genome-wide misincorporation of nonfunctional CenH3-containing nucleosomes that protect ～135 base pairs and are preferentially enriched at sites of high nucleosome turnover. Our detection of two forms of CenH3 nucleosomes in the yeast genome, a singly wrapped particle at the functional centromere and octamer-sized particles on chromosome arms, reconcile seemingly conflicting in vivo and in vitro observations.     

Nucleosome-depleted chromatin gaps recruit assembly factors for the H3.3 histone variant

Most nucleosomes that package eukaryotic DNA are assembled during DNA replication, but chromatin structure is routinely disrupted in active regions of the genome. Replication-independent nucleosome replacement using the H3.3 histone variant efficiently repackages these regions, but how histones are recruited to these sites is unknown. Here, we use an inducible system that produces nucleosome-depleted chromatin at the Hsp70 genes in Drosophila to define steps in the mechanism of nucleosome replacement. We find that the Xnp chromatin remodeler and the Hira histone chaperone independently bind nucleosome-depleted chromatin. Surprisingly, these two factors are only displaced when new nucleosomes are assembled. H3.3 deposition assays reveal that Xnp and Hira are required for efficient nucleosome replacement, and double-mutants are lethal. We propose that Xnp and Hira recognize exposed DNA and serve as a binding platform for the efficient recruitment of H3.3 predeposition complexes to chromatin gaps. These results uncover the mechanisms by which eukaryotic cells actively prevent the exposure of DNA in the nucleus.     

The structure of DNA in a nucleosome.

We describe the application of the hydroxyl radical footprinting technique to examine the histone-DNA interactions of a nucleosome that includes part of the 5S ribosomal RNA gene of Xenopus borealis. We establish that two distinct regions of DNA with different helical periodicities exist within the nucleosome and demonstrate a change in the helical periodicity of this DNA upon nucleosome formation. In particular, we find that on average the helical periodicity of DNA in this nucleosome is 10.18 +/- 0.05 base pairs per turn. The same DNA, when bound to a calcium phosphate surface, has a periodicity of 10.49 +/- 0.05 base pairs per turn, similar to that of random sequence DNA. Modulations in minor groove width within the naked DNA detected by the hydroxyl radical are maintained and exaggerated in nucleosomal DNA. These features correlate with regions in the DNA previously suggested to be important for nucleosome positioning.     

Formation of functional centromeric chromatin is specified epigenetically in Candida albicans

In the pathogenic yeast Candida albicans, the 3-kb centromeric DNA regions (CEN) of each of the eight chromosomes have different and unique DNA sequences. The centromeric histone CaCse4p (CENP-A homolog) occurs only within these 3-kb CEN regions to form specialized centromeric chromatin. Centromere activity was maintained on small chromosome fragments derived in vivo by homologous recombination of a native chromosome with linear DNA fragments containing a telomere and a selectable marker. An in vivo derived 85-kb truncated chromosome containing the 3-kb CEN7 locus on 69 kb of chromosome 7 DNA was stably and autonomously maintained in mitosis, indicating that preexisting active CEN chromatin remains functional through many generations. This same 85-kb chromosome fragment, isolated as naked DNA (devoid of chromatin proteins) from C. albicans and reintroduced back into C. albicans cells by standard DNA transformation techniques, was unable to reform functional CEN chromatin and was mitotically unstable. Comparison of active and inactive CEN chromatin digested with micrococcal nuclease revealed that periodic nucleosome arrays are disrupted at active centromeres. Chromatin immunoprecipitation with antibodies against CaCse4p confirmed that CEN7 introduced into C. albicans cells as naked DNA did not recruit CaCse4p or induce its spread to a duplicate region only 7 kb away from active CEN7 chromatin. These results indicate that CaCse4p recruitment and centromere activation are epigenetically specified and maintained in C. albicans.     

Structure of chromatin and the linking number of DNA.

Recent observations suggest that the basic supranucleosomal structure of chromatin is a zigzag helical ribbon with a repeat unit made of two nucleosomes connected by a relaxed spacer DNA. A remarkable feature of one particular ribbon is that it solves the apparent paradox between the number of DNA turns per nucleosome and the total linking number of a nucleosome-containing closed circular DNA molecule. We show here that the repeat unit of the proposed structure, which contains two nucleosomes with -1 3/4 DNA turns per nucleosome and one spacer crossover per repeat, contributes -2 to the linking number of closed circular DNA. Space-filling models show that the cylindrical 250-A chromatin fiber can be generated by twisting the ribbon.     

A chromatin folding model that incorporates linker variability generates fibers resembling the native structures.

The "30-nm" chromatin fibers, as observed in eukaryotic nuclei, are considered a discrete level in a hierarchy of DNA folding. At present, there is considerable debate as to how the nucleosomes and linker DNA are organized within chromatin fibers, and a number of models have been proposed, many of which are based on helical symmetry and imply specific contacts between nucleosomes. However, when observed in nuclei or after isolation, chromatin fibers show considerable structural irregularity. In the present study, chromatin folding is considered solely in terms of the known properties of the nucleosome-linker unit, taking into account the relative rotation between consecutive nucleosomes that results from the helical twist of DNA. Model building based on this premise, and with a constant length of linker DNA between consecutive nucleosomes, results in a family of fiber- and ribbon-like structures. When the linker length between nucleosomes is allowed to vary, as occurs in nature, fibers showing the types of irregularity observed in nuclei and in isolated chromatin are created. The potential application of the model in determining the three-dimensional organization of chromatin in which nucleosome positions are known is discussed.     

Thymine dimer formation as a probe of the path of DNA in and between nucleosomes in intact chromatin.

Photo-induced thymine dimer formation was used to probe nucleosome structure in nuclei. The distribution of thymine dimers in the nucleosome and recent studies of the structure of thymine dimer-containing DNA suggest that the rate of thymine dimer formation is affected by the direction and degree of DNA bending. This premise was used to construct a model of the path of DNA in the nucleosome, which has the following features. (i) There are four regions of sharp bending, two which have been seen previously by x-ray crystallography of the core particle. (ii) The DNA in H1-containing nucleosomes deviates from its superhelical path near the midpoint; this is not seen with H1-stripped chromatin. (iii) The internucleosomal (linker) DNA appears to be relatively straight.