Organization Of Eukaryotic DNA Research Articles

Molecular motor in a box: Attractors, repellers, and ratchets in chromatin

The spatiotemporal organization of eukaryotic DNA is controlled by an intricate combination of passive (thermally activated) and active (ATP-consuming) processes. Based on recent experimental insights into the mechanochemical cycle of chromatin remodelers, molecular machines that actively control nucleosome positions, we introduce a model to study the competition between active and passive mechanisms at the most basic layer of DNA packaging, the wrapping of DNA into nucleosomes. Depending on the level of remodeler activity, the positions of nucleosomes are controlled by either the bending or the stretching energy of the wrapped DNA involved. Since these energies are highly sequence dependent, DNA guides its own packaging. However, since this dependence differs for the two deformation modes, active processes can drive nucleosomes from their equilibrium positions. Furthermore, for repetitive DNA sequences, such as telomeres, we find thermal ratchets that propel nucleosomes in a preferred direction. Published by the American Physical Society 2024

Probing Nucleosome Stability with a DNA Origami Nanocaliper.

The organization of eukaryotic DNA into nucleosomes and chromatin undergoes dynamic structural changes to regulate genome processing, including transcription and DNA repair. Critical chromatin rearrangements occur over a wide range of distances, including the mesoscopic length scale of tens of nanometers. However, there is a lack of methodologies that probe changes over this mesoscopic length scale within chromatin. We have designed, constructed, and implemented a DNA-based nanocaliper that probes this mesoscopic length scale. We developed an approach of integrating nucleosomes into our nanocaliper at two attachment points with over 50% efficiency. Here, we focused on attaching the two DNA ends of the nucleosome to the ends of the two nanocaliper arms, so the hinge angle is a readout of the nucleosome end-to-end distance. We demonstrate that nucleosomes integrated with 6, 26, and 51 bp linker DNA are partially unwrapped by the nanocaliper by an amount consistent with previously observed structural transitions. In contrast, the nucleosomes integrated with the longer 75 bp linker DNA remain fully wrapped. We found that the nanocaliper angle is a sensitive measure of nucleosome disassembly and can read out transcription factor (TF) binding to its target site within the nucleosome. Interestingly, the nanocaliper not only detects TF binding but also significantly increases the probability of TF occupancy at its site by partially unwrapping the nucleosome. These studies demonstrate the feasibility of using DNA nanotechnology to both detect and manipulate nucleosome structure, which provides a foundation of future mesoscale studies of nucleosome and chromatin structural dynamics.

Universal internucleotide statistics in full genomes: a footprint of the DNA structure and packaging?

Uncovering the fundamental laws that govern the complex DNA structural organization remains challenging and is largely based upon reconstructions from the primary nucleotide sequences. Here we investigate the distributions of the internucleotide intervals and their persistence properties in complete genomes of various organisms from Archaea and Bacteria to H. Sapiens aiming to reveal the manifestation of the universal DNA architecture. We find that in all considered organisms the internucleotide interval distributions exhibit the same -exponential form. While in prokaryotes a single -exponential function makes the best fit, in eukaryotes the PDF contains additionally a second -exponential, which in the human genome makes a perfect approximation over nearly 10 decades. We suggest that this functional form is a footprint of the heterogeneous DNA structure, where the first -exponential reflects the universal helical pitch that appears both in pro- and eukaryotic DNA, while the second -exponential is a specific marker of the large-scale eukaryotic DNA organization.

Histone chaperones: assisting histone traffic and nucleosome dynamics.

The functional organization of eukaryotic DNA into chromatin uses histones as components of its building block, the nucleosome. Histone chaperones, which are proteins that escort histones throughout their cellular life, are key actors in all facets of histone metabolism; they regulate the supply and dynamics of histones at chromatin for its assembly and disassembly. Histone chaperones can also participate in the distribution of histone variants, thereby defining distinct chromatin landscapes of importance for genome function, stability, and cell identity. Here, we discuss our current knowledge of the known histone chaperones and their histone partners, focusing on histone H3 and its variants. We then place them into an escort network that distributes these histones in various deposition pathways. Through their distinct interfaces, we show how they affect dynamics during DNA replication, DNA damage, and transcription, and how they maintain genome integrity. Finally, we discuss the importance of histone chaperones during development and describe how misregulation of the histone flow can link to disease.

Chromatin and DNA Replication

The size of a eukaryotic genome presents a unique challenge to the cell: package and organize the DNA to fit within the confines of the nucleus while at the same time ensuring sufficient dynamics to allow access to specific sequences and features such as genes and regulatory elements. This is achieved via the dynamic nucleoprotein organization of eukaryotic DNA into chromatin. The basic unit of chromatin, the nucleosome, comprises a core particle with 147 bp of DNA wrapped 1.7 times around an octamer of histones. The nucleosome is a highly versatile and modular structure, both in its composition, with the existence of various histone variants, and through the addition of a series of posttranslational modifications on the histones. This versatility allows for both short-term regulatory responses to external signaling, as well as the long-term and multigenerational definition of large functional chromosomal domains within the nucleus, such as the centromere. Chromatin organization and its dynamics participate in essentially all DNA-templated processes, including transcription, replication, recombination, and repair. Here we will focus mainly on nucleosomal organization and describe the pathways and mechanisms that contribute to assembly of this organization and the role of chromatin in regulating the DNA replication program.

Chromatin Remodeling, DNA Damage Repair and Aging

Cells are constantly exposed to a variety of environmental and endogenous conditions causing DNA damage, which is detected and repaired by conserved DNA repair pathways to maintain genomic integrity. Chromatin remodeling is critical in this process, as the organization of eukaryotic DNA into compact chromatin presents a natural barrier to all DNA-related events. Studies on human premature aging syndromes together with normal aging have suggested that accumulated damages might lead to exhaustion of resources that are required for physiological functions and thus accelerate aging. In this manuscript, combining the present understandings and latest findings, we focus mainly on discussing the role of chromatin remodeling in the repair of DNA double-strand breaks (DSBs) and regulation of aging.

Standardized collection of MNase-seq experiments enables unbiased dataset comparisons

BackgroundThe organization of eukaryotic DNA into chromatin has a strong influence on the accessibility and regulation of genetic information. The locations and occupancies of a principle component of chromatin, nucleosomes, are typically assayed through use of enzymatic digestion with micrococcal nuclease (MNase). MNase is an endo-exo nuclease that preferentially digests naked DNA and the DNA in linkers between nucleosomes, thus enriching for nucleosome-associated DNA. To determine nucleosome organization genome-wide, DNA remaining from MNase digestion is sequenced using high-throughput sequencing technologies (MNase-seq). Unfortunately, the results of MNase-seq can vary dramatically due to technical differences and this confounds comparisons between MNase-seq experiments, such as examining condition-dependent chromatin organizations.ResultsIn this study we use MNase digestion simulations to demonstrate how MNase-seq signals can vary for different nucleosome configuration when experiments are performed with different extents of MNase digestion. Signal variation in these simulations reveals an important DNA sampling bias that results from a neighborhood effect of MNase digestion techniques. The presence of this neighborhood effect ultimately confounds comparisons between different MNase-seq experiments. To address this issue we present a standardized chromatin preparation which controls for technical variance between MNase-based chromatin preparations and enables the collection of similarly sampled (matched) chromatin populations. Standardized preparation of chromatin includes a normalization step for DNA input into MNase digestions and close matching of the extent of digestion between each chromatin preparation using gel densitometry analysis. The protocol also includes directions for successful pairing with multiplex sequencing reactions.ConclusionsWe validated our method by comparing the experiment-to-experiment variation between biological replicates of chromatin preparations from S. cerevisiae. Results from our matched preparation consistently produced MNase-seq datasets that were more closely correlated than other unstandardized approaches. Additionally, we validated the ability of our approach at enabling accurate downstream comparisons of chromatin structures, by comparing the specificity of detecting Tup1-dependent chromatin remodeling events in comparisons between matched and un-matched wild-type and tup1Δ MNase-seq datasets. Our matched MNase-seq datasets demonstrated a significant reduction in non-specific (technical) differences between experiments and were able to maximize the detection of biologically-relevant (Tup1-dependent) changes in chromatin structure.

Repertoires of the Nucleosome-Positioning Dinucleotides

It is generally accepted that the organization of eukaryotic DNA into chromatin is strongly governed by a code inherent in the genomic DNA sequence. This code, as well as other codes, is superposed on the triplets coding for amino acids. The history of the chromatin code started three decades ago with the discovery of the periodic appearance of certain dinucleotides, with AA/TT and RR/YY giving the strongest signals, all with a period of 10.4 bases. Every base-pair stack in the DNA duplex has specific deformation properties, thus favoring DNA bending in a specific direction. The appearance of the corresponding dinucleotide at the distance 10.4 xn bases will facilitate DNA bending in that direction, which corresponds to the minimum energy of DNA folding in the nucleosome. We have analyzed the periodic appearances of all 16 dinucleotides in the genomes of thirteen different eukaryotic organisms. Our data show that a large variety of dinucleotides (if not all) are, apparently, contributing to the nucleosome positioning code. The choice of the periodical dinucleotides differs considerably from one organism to another. Among other 10.4 base periodicities, a strong and very regular 10.4 base signal was observed for CG dinucleotides in the genome of the honey bee A. mellifera. Also, the dinucleotide CG appears as the only periodical component in the human genome. This observation seems especially relevant since CpG methylation is well known to modulate chromatin packing and regularity. Thus, the selection of the dinucleotides contributing to the chromatin code is species specific, and may differ from region to region, depending on the sequence context.

ATP-dependent chromatin remodeling factors and DNA damage repair

The organization of eukaryotic DNA into chromatin poses a barrier to all processes that require access of enzymes and regulatory factors to their sites of action. While the majority of studies in this area have concentrated on the role of chromatin in the regulation of transcription, there has been a recent emphasis on the relationship of chromatin to DNA damage repair. In this review, we focus on the role of chromatin in nucleotide excision repair (NER) and double-strand break (DSB) repair. NER and DSB repair use very different enzymatic machineries, and these two modes of DNA damage repair are also differentially affected by chromatin. Only a small number of nucleosomes are likely to be involved in NER, while a more extensive region of chromatin is involved in DSB repair. However, a key feature of both NER and DSB repair pathways is the participation of ATP-dependent chromatin remodeling factors at various points in the repair process. We discuss recent data that have identified roles for SWI/SNF-related chromatin remodeling factors in the two repair pathways.

Rtt109 Is Required for Proper H3K56 Acetylation

Histone acetylation has been shown to be required for the proper regulation of many cellular processes including transcription, DNA repair, and chromatin assembly. Acetylation of histone H3 on lysine 56 (H3K56) occurs both during the premeiotic and mitotic S phase and persists throughout DNA damage repair. To learn more about the molecular mechanism of H3K56 acetylation and factors required for this process, we surveyed the genome of the yeast Saccharomyces cerevisiae to identify genes necessary for this process. A comparative global proteomic screen identified several factors required for global H3K56 acetylation, which included histone chaperone Asf1 and a protein of an unknown function Rtt109 but not Spt10. Our results indicate that the loss of Rtt109 results in the loss of H3K56 acetylation, both on bulk histone and on chromatin, similar to that of asf1Delta or the K56Q mutation. RTT109 deletion exhibits sensitivity to DNA damaging agents similar to that of asf1Delta and H3K56Q mutants. Furthermore, Rtt109 and H3K56 acetylation appear to correlate with actively transcribed genes and associate with the elongating form of polymerase II in yeast. This histone modification is also associated with some of the transcriptionally active puff sites in Drosophila. Our results indicate a new role for the Rtt109 protein in the proper regulation of H3K56 acetylation.

Measurement of low-frequency DNA breaks using nucleoid flow cytometry.

The organization of eukaryotic DNA is increasingly well understood at each extreme of chromatin organization. Advances in understanding the organization of DNA at the level of nucleosome and chromosome have outstripped knowledge of the intermediate states and function of DNA. Most would, however, agree on the following general hierarchy (,). The base-repeating unit of approx 146 bp, including a core histone group, is further organized into the 30-nm fiber and constrained by contact with the nuclear matrix to form loop and superloop structures capable of maintaining physical tension or supercoiling (, , ). These supercoiled units of DNA subsequently adopt repetitive patterns based around a protein core template, which together are observed as chromosomes.

Twists and turns of the nucleosome: tails without ends.

The high-resolution structure of a nucleosome core particle gives us our first detailed look at the primary level of eukaryotic DNA organization. The structure reveals the nature of histone-DNA contacts and provides some surprises regarding the histone tails and their possible involvement in higher levels of chromatin organization.

The channels model of nuclear matrix structure

The specificity of eukaryotic DNA organization into loops fixed to the nuclear matrix/chromosomal scaffold has been studied for more than fifteen years. The results and conclusions of different authors remain, however, controversial. Recently, we have elaborated a new approach to the study of chromosomal DNA loops. Instead of characterizing loop basements (nuclear matrix DNA), we have concentrated our efforts on the characterization of individual loops after their excision by DNA topoisomerase II-mediated DNA cleavage at matrix attachment sites. In this review the results of applying this mapping approach are compared with the results and conclusions from studies of nuclear matrix DNA. An attempt is also made to reconsider all data about the specificity of DNA interactions with the nuclear matrix and to suggest a model of spatial organization of the eukaryotic genome which resolves apparent contradictions between these data.

The structure of the sleeping genome: Implications of sperm DNA organization for somatic cells

The tertiary structure of the DNA that makes up the eukaryotic genome is remarkably plastic, taking many different forms in response to the different needs of the cell. During the cell cycle of one cell, the DNA is replicated, reorganized into mitotic chromosomes, and decondensed into interphase chromatin. Within one cell at any given point in time, the chromatin is divided into hetero- and euchromatin reflecting active and inactive states of the DNA. This organization varies within one organism since different parts of the genome are active in different cell types. This article focuses on the most dramatic cell-type-specific DNA organization, that found in spermatozoa, in which the entire genome is reorganized into an inactive state that is more highly condensed than mitotic chromosomes. This unique example of eukaryotic DNA organization offers some interesting clues to the still unanswered questions about the role that the three-dimensional packaging of DNA plays in its function.

Species-specific patterns of DNA bending and sequence

Nucleotide sequences in the GenEMBL database were analyzed using strategies designed to reveal species-specific patterns of DNA bending and DNA sequence. The results uncovered striking species-dependent patterns of bending with more variations among individual organisms than between prokaryotes and eukaryotes. The frequency of bent sites in sequences from different bacteria was related to genomic A + T content and this relationship was confirmed by electrophoretic analysis of genomic DNA. However, base composition was not an accurate predictor for DNA bending in eukaryotes. Sequences from C. elegans exhibited the highest frequency of bent sites in the database and the RNA polymerase II locus from the nematode was the most bent gene in GenEMBL. Bent DNA extended throughout most introns and gene flanking segments from C.elegans while exon regions lacked A-tract bending characteristics. Independent evidence for the strong bending character of this genome was provided by electrophoretic studies which revealed that a large number of the fragments from C.elegans DNA exhibited anomalous gel mobilities when compared to genomic fragments from over 20 other organisms. The prevalence of bent sites in this genome enabled us to detect selectively C.elegans sequences in a computer search of the database using as probes C.elegans introns, bending elements, and a 20 nucleotide consensus sequence for bent DNA. This approach was also used to provide additional examples of species-specific sequence patterns in eukaryotes where it was shown that (A) greater than or equal to 10 and (A.T) greater than or equal to 5 tracts are prevalent throughout the untranslated DNA of D.discodium and P.falciparum, respectively. These results provide new insight into the organization of eukaryotic DNA because they show that species-specific patterns of simple sequences are found in introns and in other untranslated regions of the genome.

The domain model for eukaryotic DNA organization 2: A molecular basis for constraints on development and evolution

A model for eukaryotic DNA organization has been proposed in which DNA regulatory processes depend on multiple site-specific DNA-nuclear matrix interactions throughout a DNA domain. In this model gene regulation depends on combinations of a few control factors in a cell to activate cell type-specific genes. This model suggests simple molecular mechanisms for organismal development which can account for sequential activation of appropriate groups of genes throughout development and for specific constraints on developmental pathways. Additionally, these suggested developmental pathways are consistent with mechanisms of evolution in which gradualism and punctuated equilibrium are not exclusive of one another and are interrelated mechanisms of evolution that are both induced by specific chromosomal mutations.

A domain model for eukaryotic DNA organization: A molecular basis for cell differentiation and chromosome evolution

A model for eukaryotic chromatin organization is presented in which the basic structural and functional unit is the DNA domain. This simple model predicts that both chromosome replication and cell type-specific control of gene expression depend on a combination of stable and dynamic DNA-nuclear matrix interactions. The model suggests that in eukaryotes, DNA regulatory processes are controlled mainly by the intranuclear compartmentalization of the specific DNA sequences, and that control of gene expression involves multiple steps of specific DNA-nuclear matrix interactions. Predictions of the model are tested using available biochemical, molecular and cell biological data. In addition, the domain model is discussed as a simple molecular mechanism to explain cell differentiation in multi-cellular organisms and to explain the evolution of eukaryotic genomes consisting mainly of repetitive sequences and "junk" DNA.

Structural organization of DNA

This chapter summarizes (a) the building blocks of nucleotides and in this context discusses separately the nucleic acid bases, the sugar and phosphate components, (b) the bond types in nucleotides, (c) the metabolism of nucleotides, (d) the lower levels of DNA structure (primary, secondary, tertiary, nucleosomal), (e) supranucleosomal organization of eukaryotic DNA, (f) chromosomes of animal cells, and (g) the temporal and spatial order of gene replication.

Hybridization of nuclear matrix attached deoxyribonucleic acid fragments.

Annealing studies were performed on DNA fragments associated with rat and mouse liver interphase nuclear matrix and the metaphase scaffold of Chinese hamster DON cells. Matrix and scaffold bound DNA fragments, reassociated with an excess of total genomic DNA, displayed kinetics virtually identical with total nuclear DNA probes. Moreover, both the extent and kinetics of these hybridizations were independent of the matrix DNA fragment size (less than 350--5000 base pairs) and the method of nuclease digestion used in their preparation (DNase I, micrococcal nuclease or endogenous digestion). The repetitive DNA component of the matrix DNA was examined by reacting discrete sizes of matrix DNA fragments (less than 350--5000 base pairs) from mouse liver with a library of cloned repetitive sequence DNA fragments which included mouse major satellite sequences. Our results demonstrate that short DNA fragments anchored to the nuclear matrix contain these cloned sequences is similar proportion of total nuclear DNA and, when viewed in light of the annealing results, indicate that matrix DNA is not enriched in either repetitive or unique sequences. Furthermore, the matrix DNA fragments appear to contain the entire sequence complexity of the genome. Finally, we hybridized both matrix and total nuclear DNA fragments with cDNA to total nuclear polyadenylated RNA. The kinetics and extent of hybridization indicate that most, if not all, of the actively transcribed DNA sequences are present in similar concentrations. We conclude that in the overall organization of eukaryotic DNA within the nucleus, the repeating domains or loops which have been demonstrated by a number of investigators are not anchored at specific attachment sequences in interphase cells or during mitosis. These findings are discussed with regard to current concepts of eukaryotic DNA loop organization.