Real-time detection of single-molecule DNA compaction by condensin I

BACKGROUND: Condensin is thought to contribute to large-scale DNA compaction during mitotic chromosome assembly. It remains unknown, however, how the complex reconfigures DNA structure at a mechanistic level. RESULTS: We have performed single-molecule DNA nanomanipulation experiments to directly measure in real-time DNA compaction by the Xenopus laevis condensin I complex. Condensin can bind to the nanomanipulated DNA in the absence of ATP, but it compacts the DNA only in the presence of hydrolyzable ATP. Linear compaction is evidenced by a reduction in the end-to-end extension of nanomanipulated DNA. The reaction results in total compaction of the DNA (i.e., zero end-to-end extension). Discrete and reversible DNA compaction events are observed in the presence of competitor DNA when the DNA is subjected to weak stretching forces (F = 0.4 picoNewton [pN]). The distribution of step sizes is broad and displays a peak at approximately 60 nm ( approximately 180 bp) as well as a long tail. This distribution is essentially unaffected by the topological state of the DNA substrate. Increasing the force to F = 10 pN drives the system toward step-wise reversal of compaction. The distribution of step sizes observed upon disruption of condensin-DNA interactions displays a sharp peak at approximately 30 nm ( approximately 90 bp) as well as a long tail stretching out to hundreds of nanometers. CONCLUSIONS: The DNA nanomanipulation assay allows us to demonstrate for the first time that condensin physically compacts DNA in an ATP-hydrolysis-dependent manner. Our results suggest that the condensin complex may induce DNA compaction by dynamically and reversibly introducing loops along the DNA.     

DNA supercoiling by gyrase is linked to nucleoid compaction

The genes of E. coli are located on a circular chromosome of 4.6 million basepairs. This 1.6 mm long molecule is compressed into a nucleoid to fit inside the 1-2 m cell in a functional format. To examine the role of DNA supercoiling as nucleoid compaction force we modulated the activity of DNA gyrase by electronic, genetic, and chemical means. A model based on physical properties of DNA and other cell components predicts that relaxation of supercoiling expands the nucleoid. Nucleoid size did not increase after reduction of DNA gyrase activity by genetic or chemical means, but nucleoids did expand upon chemical inhibition of gyrase in chloramphenicol-treated cells, indicating that supercoiling may help to compress the genome.     

H-NS mediated compaction of DNA visualised by atomic force microscopy

The Escherichia coli H-NS protein is a nucleoid-associated protein involved in gene regulation and DNA compaction. To get more insight into the mechanism of DNA compaction we applied atomic force microscopy (AFM) to study the structure of H-NS–DNA complexes. On circular DNA molecules two different levels of H-NS induced condensation were observed. H-NS induced lateral condensation of large regions of the plasmid. In addition, large globular structures were identified that incorporated a considerable amount of DNA. The formation of these globular structures appeared not to be dependent on any specific sequence. On the basis of the AFM images, a model for global condensation of the chromosomal DNA by H-NS is proposed.     

DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels

The Cox protein from bacteriophage P2 forms oligomeric filaments and it has been proposed that DNA can be wound up around these filaments, similar to how histones condense DNA. We here use fluorescence microscopy to study single DNA–Cox complexes in nanofluidic channels and compare how the Cox homologs from phages P2 and WΦ affect DNA. By measuring the extension of nanoconfined DNA in absence and presence of Cox we show that the protein compacts DNA and that the binding is highly cooperative, in agreement with the model of a Cox filament around which DNA is wrapped. Furthermore, comparing microscopy images for the wild-type P2 Cox protein and two mutants allows us to discriminate between compaction due to filament formation and compaction by monomeric Cox. P2 and WΦ Cox have similar effects on the physical properties of DNA and the subtle, but significant, differences in DNA binding are due to differences in binding affinity rather than binding mode. The presented work highlights the use of single DNA molecule studies to confirm structural predictions from X-ray crystallography. It also shows how a small protein by oligomerization can have great impact on the organization of DNA and thereby fulfill multiple regulatory functions.     

Kid-mediated chromosome compaction ensures proper nuclear envelope formation

Toward the end of mitosis, neighboring chromosomes gather closely to form a compact cluster. This is important for reassembling the nuclear envelope around the entire chromosome mass but not individual chromosomes. By analyzing mice and cultured cells lacking the expression of chromokinesin Kid/kinesin-10, we show that Kid localizes to the boundaries of anaphase and telophase chromosomes and contributes to the shortening of the anaphase chromosome mass along the spindle axis. Loss of Kid-mediated anaphase chromosome compaction often causes the formation of multinucleated cells, specifically at oocyte meiosis II and the first couple of mitoses leading to embryonic death. In contrast, neither male meiosis nor somatic mitosis after the morula-stage is affected by Kid deficiency. These data suggest that Kid-mediated anaphase/telophase chromosome compaction prevents formation of multinucleated cells. This protection is especially important during the very early stages of development, when the embryonic cells are rich in ooplasm.     

DNA methylation affects nuclear organization, histone modifications, and linker histone binding but not chromatin compaction

DNA methylation has been implicated in chromatin condensation and nuclear organization, especially at sites of constitutive heterochromatin. How this is mediated has not been clear. In this study, using mutant mouse embryonic stem cells completely lacking in DNA methylation, we show that DNA methylation affects nuclear organization and nucleosome structure but not chromatin compaction. In the absence of DNA methylation, there is increased nuclear clustering of pericentric heterochromatin and extensive changes in primary chromatin structure. Global levels of histone H3 methylation and acetylation are altered, and there is a decrease in the mobility of linker histones. However, the compaction of both bulk chromatin and heterochromatin, as assayed by nuclease digestion and sucrose gradient sedimentation, is unaltered by the loss of DNA methylation. This study shows how the complete loss of a major epigenetic mark can have an impact on unexpected levels of chromatin structure and nuclear organization and provides evidence for a novel link between DNA methylation and linker histones in the regulation of chromatin structure.     

Purification of plasmid DNA using selective precipitation by compaction agents.

A scaleable method for the liquid-phase separation of plasmid DNA from RNA.     

SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.     

Attenuation of DNA charge transport by compaction into a nucleosome core particle

The nucleosome core particle (NCP) is the fundamental building block of chromatin which compacts ~146 bp of DNA around a core histone protein octamer. The effects of NCP packaging on long-range DNA charge transport reactions have not been adequately assessed to date. Here we study DNA hole transport reactions in a 157 bp DNA duplex (AQ-157TG) incorporating multiple repeats of the DNA TG-motif, a strong NCP positioning sequence and a covalently attached Anthraquinone photooxidant. Following a thorough biophysical characterization of the structure of AQ-157TG NCPs by Exonuclease III and hydroxyl radical footprinting, we compared the dynamics of DNA charge transport in ultraviolet-irradiated free and NCP-incorporated AQ-157TG. Compaction into a NCP changes the charge transport dynamics in AQ-157TG drastically. Not only is the overall yield of oxidative lesions decreased in the NCPs, but the preferred sites of oxidative damage change as well. This NCP-dependent attenuation of DNA charge transport is attributed to DNA–protein interactions involving the folded histone core since removal of the histone tails did not perturb the charge transport dynamics in AQ-157TG NCPs.     

DNA end-joining driven by microhomologies catalyzed by nuclear extracts

In a previous work we used an in vitro system for the generation and analysis of double-strand breaks (DSBs) using nuclear extracts from rat testes as a source of DSB activity. Since the recombination process can be triggered by the formation of DSB, in the present study we developed a strategy to isolate and characterize recombinant molecules using the same in vitro system. Our results indicate that the mechanism for the formation of recombinants was non-homologous end-joining driven by microhomologies. The procedure described here represents an alternative to investigate the mechanisms of DNA end-joining and other forms of DNA repair.     

Effects of Hfq on the conformation and compaction of DNA

Hfq is a bacterial pleiotropic regulator that mediates several aspects of nucleic acids metabolism. The protein notably influences translation and turnover of cellular RNAs. Although most previous contributions concentrated on Hfq''s interaction with RNA, its association to DNA has also been observed in vitro and in vivo. Here, we focus on DNA-compacting properties of Hfq. Various experimental technologies, including fluorescence microscopy imaging of single DNA molecules confined inside nanofluidic channels, atomic force microscopy and small angle neutron scattering have been used to follow the assembly of Hfq on DNA. Our results show that Hfq forms a nucleoprotein complex, changes the mechanical properties of the double helix and compacts DNA into a condensed form. We propose a compaction mechanism based on protein-mediated bridging of DNA segments. The propensity for bridging is presumably related to multi-arm functionality of the Hfq hexamer, resulting from binding of the C-terminal domains to the duplex. Results are discussed in regard to previous results obtained for H-NS, with important implications for protein binding related gene regulation.     

Global chromatin compaction limits the strength of the DNA damage response

In response to DNA damage, chromatin undergoes a global decondensation process that has been proposed to facilitate genome surveillance. However, the impact that chromatin compaction has on the DNA damage response (DDR) has not directly been tested and thus remains speculative. We apply two independent approaches (one based on murine embryonic stem cells with reduced amounts of the linker histone H1 and the second making use of histone deacetylase inhibitors) to show that the strength of the DDR is amplified in the context of "open" chromatin. H1-depleted cells are hyperresistant to DNA damage and present hypersensitive checkpoints, phenotypes that we show are explained by an increase in the amount of signaling generated at each DNA break. Furthermore, the decrease in H1 leads to a general increase in telomere length, an as of yet unrecognized role for H1 in the regulation of chromosome structure. We propose that slight differences in the epigenetic configuration might account for the cell-to-cell variation in the strength of the DDR observed when groups of cells are challenged with DNA breaks.     

Chromosome condensation: DNA compaction in real time

Mitotic chromosomes must be organised into a highly ordered and compacted form to allow proper segregation of DNA during each round of cell division. Two new studies report observations of DNA compaction by eukaryotic and bacterial condensin molecules in real time using magnetic and optical trapping micromanipulation techniques.