Chromatin In Eukaryotic Cells Research Articles

Implication of Posttranslational Histone Modifications in Nucleotide Excision Repair

Histones are highly alkaline proteins that package and order the DNA into chromatin in eukaryotic cells. Nucleotide excision repair (NER) is a conserved multistep reaction that removes a wide range of generally bulky and/or helix-distorting DNA lesions. Although the core biochemical mechanism of NER is relatively well known, how cells detect and repair lesions in diverse chromatin environments is still under intensive research. As with all DNA-related processes, the NER machinery must deal with the presence of organized chromatin and the physical obstacles it presents. A huge catalogue of posttranslational histone modifications has been documented. Although a comprehensive understanding of most of these modifications is still lacking, they are believed to be important regulatory elements for many biological processes, including DNA replication and repair, transcription and cell cycle control. Some of these modifications, including acetylation, methylation, phosphorylation and ubiquitination on the four core histones (H2A, H2B, H3 and H4) or the histone H2A variant H2AX, have been found to be implicated in different stages of the NER process. This review will summarize our recent understanding in this area.

DNA/Polymeric Micelle Self‐Assembly Mimicking Chromatin Compaction

Through self-assembly, a large variety of nanomaterials have been fabricated, which are useful in many applied and research fields. However, broader development of nanoscience and nanotechnology requires more complex nanostructures whose fabrication requires precise control of morphology, structural parameters, and distributions of components within the nanomaterials, which remains a great challenge for nanotechnology. Nature is masterful at building nanostructures with great complexity by precisely controlling multi-stage self-assembly processes. One example is the self-assembly of genomic DNA and histone octamers into chromatin in eukaryotic cells. Histone octamers are discshaped nanostructures with positively-charged binding sites specifically distributed on the edge of the disc. In the first stage, a DNA chain organizes histone octamers into a beadson-a-string structure (10 nm chromatin fiber); the bead is a nucleosome composed of one DNA segment wrapping around the edge of one histone octamer, and between neighboring beads is a short DNA linker. In the second stage, histone octamers that are preorganized on the 10 nm chromatin fiber, self-assemble further in a zigzag manner into the 30 nm chromatin fiber with a two-start helical structure. Histone octamers do not self-assemble into the 30 nm fiber until they are preorganized by the first stage of self-assembly; the first stage self-assembly initiates the second stage self-assembly. Inspired by such efficient self-assembly processes from nature, a number of DNA/sphere systems were studied; artificial nanospheres were used in the place of histone octamers. Computer simulation of the interaction between a polyelectrolyte with long DNA-like semi-flexible chains and the spheres with a uniform oppositely-charged surface reveals that the beads-on-a-string structure, similar to the 10 nm chromatin fiber, is the thermodynamic favored structure. However, experimentally, other kinetically trapped structures were inevitably produced in the reported systems because the DNA/sphere interactions were relatively strong. Herein, we prepared thermodynamically optimal structures of a DNA/artificial particle complex by controlling the interaction between DNA and the particles. We selected the block-copolymer micelles with an inert shell and a positively charged core to interact with DNA (Figure 1A); the interaction strength between DNA and the core can be continuously adjusted by the pH value of the medium. It was found that under certain conditions the strings (that are similar to the 10 nm chromatin fibers in both morphology and structure) formed exclusively. Under such conditions, only the thermodynamically favored beads-on-a-string structure can persist, while any kinetically trapped structures cannot. When monodisperse DNA was used, the strings formed were monodisperse, and the earlier-formed long strings evolved to shorter, but also monodisperse, strings; the different strings formed and evolved in a similar manner. Then, in a second stage, the micelles preorganized in the beads-on-a-string structure self-assembled along the strings into core–shell structured solenoidal nanofibers. The preorganization induced and guided the second stage of self-assembly. When monodisperse DNAs are used, the resulting nanostructures are monodisperse. This self-assembly process can be used for synthesis of monodisperse one-dimensional nanostructures with controlled dimensions and various compositions. Poly(ethylene glycol)113-b-poly(4-vinylpyridine)58 (PEG113b-P4VP58, subscripts represent the average degrees of polymerization;Mw/Mn= 1.20) micelles were prepared in a water/ methanol (4:1, v/v) mixture (see Supporting Information, Text S1). The micelles were monodisperse in size with an average hydrodynamic radius hRhi of 16.0 nm, a polydispersity index (PDI) of 0.05, and an average molecular weight of 1.74 10 gmol , based on light scattering (LS) measurements. In the TEM images, the micelles are monodisperse with a size of 17.5 1.5 nm (Figure 1B). The micelles shown have PEG as the shell and P4VP as the core. An aqueous solution of monodisperse linear doublestrandedDNA 5427 bp long (L5427) at 25 8Cwas added to the micelles in the water/methanol mixture to give a DNA/ micelle mass ratio of 1:20. The solution had a pH value of 6.6 in the presence of carbon dioxide (see Experimental Section), which provided the proper strength for the electrostatic interaction between DNA and the micelles (Supporting Information, Text S2). After 0.5 hours incubation, strings with a beads-on-a-string structure formed exclusively (Figure 1C). These strings have a linear structure without any branches, indicating that each string is composed of a single L5427 DNA chain, as detailed below. For each string, the beads were very similar in shape and size to the micelles. Between neighboring beads was a linker, which appears to be a short DNA segment. Additionally, the strings were shown to [*] K. Zhang, Prof. M. Jiang, Prof. D. Chen The State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan University Handan Road 220, Shanghai 200433 (P.R. China) E-mail: chendy@fudan.edu.cn

Predicting Nucleosome Positions in Yeast: Using the Absolute Frequency

Nucleosome is the basic structure of chromatin in eukaryotic cells, and they form the chromatin fiber interconnected by sections of linker DNA. Nucleosome positioning is of great significance for gene transcription regulation. In this paper, we consider the difference of absolute frequency of nucleotides between the nucleosome forming and nucleosome inhibiting sequences. Based on the 2-mer absolute frequency of nucleotides in genome, a new model is constructed to distinguish nucleosome DNA and linker DNA. When used to predict DNA potential for forming nucleosomes in S. cerevisiae, the model achieved a high accuracy of 96.05%. Thus, the model is very useful for predicting nucleosome positioning.

Diverse Roles of RNA Polymerase II-associated Factor 1 Complex in Different Subpathways of Nucleotide Excision Repair

Transcription-coupled repair (TCR) and global genomic repair (GGR) are two pathways of nucleotide excision repair (NER). In Saccharomyces cerevisiae, Rad26 is important but not absolutely required for TCR. Rpb4, a nonessential RNA polymerase II (Pol II) subunit that forms a subcomplex with Rpb7, and the Spt4-Spt5 complex, a transcription elongation factor, have been shown to suppress Rad26-independent TCR. The Pol II-associated factor 1 complex (Paf1C) has been shown to function in transcription elongation, 3'-processing of mRNAs, and posttranslational modification of histones. Here we show that Paf1C plays a marginal role in facilitating Rad26-dependent TCR but significantly suppresses Rad26-independent TCR. The suppression of Rad26-independent TCR is achieved by cooperating with Spt4-Spt5. We propose a model that, in the absence of Rad26, a lesion is "locked" in the active center of a Pol II elongation complex, which is stabilized by the coordinated interactions of Rpb4-Rpb7, Spt4-Spt5, and Paf1C with each other and with the core Pol II. We also found that Paf1C facilitates GGR, especially in internucleosomal linker regions. The facilitation of GGR is achieved through enabling monoubiquitination of histone H2B lysine 123 by Bre1, which in turn permits di- and trimethylation of histone H3 lysine 79 by Dot1. To our best knowledge, among the NER-modulating factors documented so far, Paf1C appears to have the most diverse functions in different NER pathways or subpathways.

The role of FACT in making and breaking nucleosomes

FACT is a roughly 180kDa heterodimeric protein complex important for managing the properties of chromatin in eukaryotic cells. Chromatin is a repressive barrier that plays an important role in protecting genomic DNA and regulating access to it. This barrier must be temporarily removed during transcription, replication, and repair, but it also must be rapidly restored to the original state afterwards. Further, the properties of chromatin are dynamic and must be adjusted as conditions dictate. FACT was identified as a factor that destabilizes nucleosomes in vitro, but it has now also been implicated as a central factor in the deposition of histones to form nucleosomes, as an exchange factor that swaps the histones within existing nucleosomes for variant forms, and as a tether that prevents histones from being displaced by the passage of RNA polymerases during transcription. FACT therefore plays central roles in building, maintaining, adjusting, and overcoming the chromatin barrier. This review summarizes recent results that have begun to reveal how FACT can promote what appear to be contradictory goals, using a simple set of binding activities to both enhance and diminish the stability of nucleosomes. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.

Evidence That the Histone Methyltransferase Dot1 Mediates Global Genomic Repair by Methylating Histone H3 on Lysine 79

Global genomic repair (GGR) and transcription coupled repair (TCR) are two pathways of nucleotide excision repair (NER) that differ in the damage recognition step. How NER factors, especially GGR factors, access DNA damage in the chromatin of eukaryotic cells has been poorly understood. Dot1, a histone methyltransferase required for methylation of histone H3 lysine 79 (H3K79), has been shown to confer yeast cells with resistance to DNA-damaging agents and play a role in activation of DNA damage checkpoints. Here, we show that Dot1 and H3K79 methylation are required for GGR in both nucleosomal core regions and internucleosomal linker DNA, but play no role in TCR. H3K79 trimethylation contributes to but is not absolutely required for GGR, and lower levels of H3K79 methylation (mono- and dimethylation) also promote GGR. Our results also indicate that the roles of Dot1 and H3K79 methylation in GGR are not achieved by either activating DNA damage checkpoints or regulating the expression of the GGR-specific factor Rad16. Rather, the methylated H3K79 may serve as a docking site for the GGR machinery on the chromatin. Our studies identified a novel GGR-specific NER factor and unveiled the critical link between a covalent histone modification and GGR.

히스톤 H3K27 변형과 유전자 전사

진핵세포의 크로마틴에서 히스톤 단백질 H3와 H4의 라이신 잔기는 공유결합에 의해 변형된다. 히스톤 H3에서 27번 라이신은 아세틸화되거나(H3K27ac) 세 가지 단계로 메틸화가 될 수 있으며(H3K27me1, H3K27me2, H3K27me3), 이러한 H3K27의 변형들은 각각 독특한 형태로 유전자 전사 및 크로마틴 구조와 관련된다. 일반적으로 H3K27ac과 H3K27me1은 좌위조절부위나 활발히 전사되는 유전자처럼 활성 크로마틴에서 나타나고, 이에 반해 전사가 일어나지 않은 유전자는 높은 수준의 H3K27me2과 H3K27me3이 관찰된다. 이러한 변형들은 각각 다른 종류의 변형효소에 의해 촉매된다. 최근 연구들은 유전자 전사 및 크로마틴 구조 형성에서 H3K27의 네 가지 변형들 사이에 상관 관계가 있음을 제시하고 있다. Lysine residues of histone H3 and H4 are covalently modified in the chromatin of eukaryotic cells. Lysine 27 in histone H3 was acetylated (H3K27ac) or methylated at three levels; mono-, di-, and trimethylation (H3K27me1, H3K27me2, and H3K27me3). These modifications at H3K27 were related with gene transcription and/or chromatin structure in distinct patterns. Generally, H3K27ac and H3K27me1 were enriched in active chromatin, such as the locus control region or transcriptionally active genes, while transcriptionally inactive genes were highly marked by H3K27me2 and H3K27me3. These modifications appear to have been catalyzed by distinct histone-modifying enzymes. Recent studies suggest that the four kinds of modifications at H3K27 have inter-correlation in gene transcription or chromatin structure formation.

Isolation of active regulatory elements from eukaryotic chromatin using FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements)

The binding of sequence-specific regulatory factors and the recruitment of chromatin remodeling activities cause nucleosomes to be evicted from chromatin in eukaryotic cells. Traditionally, these active sites have been identified experimentally through their sensitivity to nucleases. Here we describe the details of a simple procedure for the genome-wide isolation of nucleosome-depleted DNA from human chromatin, termed FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements). We also provide protocols for different methods of detecting FAIRE-enriched DNA, including use of PCR, DNA microarrays, and next-generation sequencing. FAIRE works on all eukaryotic chromatin tested to date. To perform FAIRE, chromatin is crosslinked with formaldehyde, sheared by sonication, and phenol–chloroform extracted. Most genomic DNA is crosslinked to nucleosomes and is sequestered to the interphase, whereas DNA recovered in the aqueous phase corresponds to nucleosome-depleted regions of the genome. The isolated regions are largely coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, enhancers, insulators, and active promoters. Given its speed and simplicity, FAIRE has utility in establishing chromatin profiles of diverse cell types in health and disease, isolating DNA regulatory elements en masse for further characterization, and as a screening assay for the effects of small molecules on chromatin organization.

The molecular basis of chromatin dynamics during nucleotide excision repairThis paper is one of a selection of papers published in this Special Issue, entitled 29th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process.

The assembly of DNA into chromatin in eukaryotic cells affects all DNA-related cellular activities, such as replication, transcription, recombination, and repair. Rearrangement of chromatin structure during nucleotide excision repair (NER) was discovered more than 2 decades ago. However, the molecular basis of chromatin dynamics during NER remains undefined. Pioneering studies in the field of gene transcription have shown that ATP-dependent chromatin-remodeling complexes and histone-modifying enzymes play a critical role in chromatin dynamics during transcription. Similarly, recent studies have demonstrated that the SWI/SNF chromatin-remodeling complex facilitates NER both in vitro and in vivo. Additionally, histone acetylation has also been linked to the NER of ultraviolet light damage. In this article, we will discuss the role of these identified chromatin-modifying activities in NER.

Brownian Dynamics Simulation of DNA Unrolling from the Nucleosome

Nucleosomes organize chromatin in eukaryotic cells at the lowest scale by wrapping the DNA double helix around a histone octamer. The mechanism by which this structure can be opened, giving access to DNA-processing enzymes, is of fundamental biological importance. Here we describe a new coarse-grained model based on the toroidal geometry of the nucleosome which allows the simulation of nucleosome stretching experiments with a Brownian dynamics algorithm including hydrodynamics. We obtain force-extension curves and calculate energy barriers and kinetic rate constants of the unrolling transition from rupture forces.

New insights into the chromosome cycle

The EMBO Conference on the Replication & Segregation of Chromosomes took place between 16 and 20 June 2008, in Geilo, Norway, and was organized by E. Boye & K. Skarstad ![][1] The mechanisms by which cells replicate and segregate their chromosomes have been the subject of intense study for several decades, yet many fundamental questions still remain unanswered. This is partly due to the complexity of these processes, which are regulated so that they are tightly coupled to cell growth and cell division, and have an extremely low error rate, thus allowing the stable inheritance of the genome. The elaborate nature of chromatin in eukaryotic cells further complicates the process of chromosome duplication, as the epigenetic patterns of histone modifications throughout the genome must also be copied faithfully. Moreover, chromosome replication is coupled to other processes such as the establishment of cohesion between the resultant sister chromatids, which facilitates mitosis and promotes the repair of DNA breaks by homologous recombination (Fig 1). Many of these processes have not yet been reconstituted using purified proteins, and much remains to be discovered. The EMBO meeting on the Replication & Segregation of Chromosomes in Geilo, Norway, brought together people working on these issues in diverse eukaryotic and prokaryotic species. Here, we provide an overview of some meeting highlights, but space constraints prevent us from covering all of the talks. We therefore apologize to those whose work we have not been able to discuss. Figure 1. The chromosome cycle in bacteria and eukaryotes. Replication of the circular chromosome of Escherichia coli starts at a single origin and the segregation of sister chromosomes takes place as replication proceeds. In eukaryotes, replication starts at many origins per linear chromosome and cohesin (orange rings) maintains sister chromatid attachment during the process. Chromosome segregation only occurs after cohesin is cleaved in … [1]: /embed/graphic-1.gif

Different structural states in oligonucleosomes are required for early versus late steps of base excision repair

Chromatin in eukaryotic cells is folded into higher order structures of folded nucleosome filaments, and DNA damage occurs at all levels of this structural hierarchy. However, little is known about the impact of higher order folding on DNA repair enzymes. We examined the catalytic activities of purified human base excision repair (BER) enzymes on uracil-containing oligonucleosome arrays, which are folded primarily into 30 nm structures when incubated in repair reaction buffers. The catalytic activities of uracil DNA glycosylase (UDG) and apyrimidinic/apurinic endonuclease (APE) digest G:U mismatches to completion in the folded oligonucleosomes without requiring significant disruption. In contrast, DNA polymerase β (Pol β) synthesis is inhibited in a major fraction (∼80%) of the oligonucleosome array, suggesting that single strand nicks in linker DNA are far more accessible to Pol β in highly folded oligonucleosomes. Importantly, this barrier in folded oligonucleosomes is removed by purified chromatin remodeling complexes. Both ISW1 and ISW2 from yeast significantly enhance Pol β accessibility to the refractory nicked sites in oligonucleosomes. These results indicate that the initial steps of BER (UDG and APE) act efficiently on highly folded oligonucleosome arrays, and chromatin remodeling may be required for the latter steps of BER in intact chromatin.

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.

Nucleosomes and regulation of gene expression. Structure of the HIV-1 5'LTR.

Packaging of DNA into chromatin adds complexity to the problem of regulation of gene expression. Nucleosomes affect the accessibility of transcription factors to occupy their binding sites in chromatin of eukaryotic cells. The disruption of nucleosome structure within the enhancer/promoter region of the integrated HIV-1 proviral genome is an instructive example of a chromatin remodeling process during transcriptional activation. To investigate the mechanism responsible for generating nuclease hypersensitive sites that exist in vivo in the promoter/enhancer region of the 5'LTR (long terminal repeat) of integrated HIV-1 we have utilized an in vitro chromatin assembly system with Xenopus oocyte extracts. Chromatin assembly in the presence of Sp1 and NFkappaB transcription factors induces DNase I hypersensitive sites on either side of their binding sites and positions the adjacent nucleosomes. This structure can also be formed in a factor-induced, ATP-dependent chromatin remodeling process and closely resembles the in vivo chromatin structure. The DNase I hypersensitive sites that form within the HIV LTR are probably histone-free and remain after removal of transcription factors.

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.

Alterations in the internucleosomal DNA helical twist in chromatin of human erythroleukemia cells in vivo influences the chromatin higher-order folding

In the present study chloroquine diphosphate, a DNA intercalating drug, was used to alter the internucleosomal DNA helical twist in chromatin of living mammalian cells. The intercalative binding of chloroquine effectively unwinds the DNA double helix and its binding is restricted to nucleosomal linker regions without noticeable disruption of nucleosomes. The results presented here imply that the alterations in the rotation angle between the adjacent nucleosomes in chromatin of eukaryotic cells in vivo significantly influences the way the chain of nucleosomes folds in higher-order chromatin structures, as evidenced by specific alterations in nuclease susceptibility of chromatin.

Studies of nucleosome structure.

The nucleosome is the universal protein/DNA element repeated throughout the chromatin of eukaryotic cells (Kornberg 1977; McGhee and Felsenfeld 1980). Presumably as a consequence of its fundamental role in the organization of DNA in a compact unit, it has also been found to take part in gene regulation (see, e.g., Pederson et al. 1986; van Holde 1988; Grunstein 1990; Wolffe 1991; Kornberg and Lorch 1992). A major objective of our laboratory has been to obtain structural information at high resolution for the nucleosome, its higher-order structure, and its interactions with other nuclear factors. Previously, the structure of the nucleosome core particle was solved by X-ray crystallography at 7 Å resolution using the method of multiple isomorphous replacement (Richmond et al. 1984). This X-ray structure confirmed the size and shape of the particle deduced earlier from several lines of information (Finch et al. 1979), showing that the “146”-bp DNA is arranged...

The multiple activities of Escherichia coli endonuclease IV and the extreme lability of 5′-terminal base-free deoxyribose 5-phosphates

Escherichia coli endonuclease IV hydrolyses the C(3')-O-P bond 5' to a 3'-terminal base-free deoxyribose. It also hydrolyses the C(3')-O-P bond 5' to a 3'-terminal base-free 2',3'-unsaturated sugar produced by nicking 3' to an AP (apurinic or apyrimidinic) site by beta-elimination; this explains why the unproductive end produced by beta-elimination is converted by the enzyme into a 3'-OH end able to prime DNA synthesis. The action of E. coli endonuclease IV on an internal AP site is more complex: in a first step the C(3')-O-P bond 5' to the AP site is hydrolysed, but in a second step the 5'-terminal base-free deoxyribose 5'-phosphate is lost. This loss is due to a spontaneous beta-elimination reaction in which the enzyme plays no role. The extreme lability of the C(3')-O-P bond 3' to a 5'-terminal AP site contrasts with the relative stability of the same bond 3' to an internal AP site; in the absence of beta-elimination catalysts, at 37 degrees C the half-life of the former is about 2 h and that of the latter 200 h. The extreme lability of a 5'-terminal AP site means that, after nicking 5' to an AP site with an AP endonuclease, in principle no 5'----3' exonuclease is needed to excise the AP site: it falls off spontaneously. We have repaired DNA containing AP sites with an AP endonuclease (E. coli endonuclease IV or the chromatin AP endonuclease from rat liver), a DNA polymerase devoid of 5'----3' exonuclease activity (Klenow polymerase or rat liver DNA polymerase beta) and a DNA ligase. Catalysts of beta-elimination, such as spermine, can drastically shorten the already brief half-life of a 5'-terminal AP site; it is what very probably happens in the chromatin of eukaryotic cells. E. coli endonuclease IV also probably participates in the repair of strand breaks produced by ionizing radiations: as E. coli endonuclease VI/exonuclease III, it is a 3'-phosphoglycollatase and also a 3'-phosphatase. The 3'-phosphatase activity of E. coli endonuclease VI/exonuclease III and E. coli endonuclease IV can also be useful when the AP site has been excised by a beta delta-elimination reaction.

N-Acetoxy-acetylaminofluorene reacts preferentially with a control region of intracellular SV40 chromosome.

Many chemical carcinogens or their metabolites react with DNA; thus it is of interest to determine what effect chromosomal structure has on these reactions. The chromosome of simian virus 40 (SV40) is well suited for such studies; like chromatin of eukaryotic cells, it is organized into nucleosomes. The nucleotide sequence of SV40 is known, together with much about the pattern of viral gene expression and DNA replication, and the structure of the viral chromosome. We have investigated the binding of the ultimate carcinogen, N-acetoxy-acetylaminofluorene (AAAF), to specific regions of the SV40 chromosome in situ in the intact infected cell. The results, reported here, indicate that a region containing regulatory functions on the intracellular SV40 chromosome has unique structural properties which render it more susceptible to attack by AAAF than the rest of the SV40 genome. The preferential binding of AAAF to regulatory regions of chromatin may have implications for the mechanism of action of this and similar carcinogens.

Complete in vitro replication of SV40 DNA and chromatin in saponin-treated permeable cells.

A permeable cell system has been developed by treatment with saponin for studying in vitro replication of DNA and chromatin. DNA replication of simian virus 40 nucleoprotein complexes (SV40 chromatin) in saponin-treated permeable cells was found to be more efficient than that in digitonin-treated permeable cells. Autoradiography of the agarose-gel revealed that [alpha-32P]dCTP was incorporated into SV40 DNA I, II and replicating intermediates. The time course of the incorporation indicated complete replication of SV40 DNA and chromatin with a full number of nucleosomes. The saponin-treated permeable cell system will serve as a useful system for studying in vitro replication of DNA and chromatin in eukaryotic cells.