The human Rad50/Mre11/Nbs1 complex (hR/M/N) functions as an essential guardian of genome integrity by directing the proper processing of DNA ends, including DNA breaks. This biological function ...results from its ability to tether broken DNA molecules. hR/M/N's dynamic molecular architecture consists of a globular DNA-binding domain from which two 50-nm-long coiled coils protrude. The coiled coils are flexible and their apices can self-associate. The flexibility of the coiled coils allows their apices to adopt an orientation favourable for interaction. However, this also allows interaction between the tips of two coiled coils within the same complex, which competes with and frustrates the intercomplex interaction required for DNA tethering. Here we show that the dynamic architecture of hR/M/N is markedly affected by DNA binding. DNA binding by the hR/M/N globular domain leads to parallel orientation of the coiled coils; this prevents intracomplex interactions and favours intercomplex associations needed for DNA tethering. The hR/M/N complex thus is an example of a biological nanomachine in which binding to its ligand, in this case DNA, affects the functional conformation of a domain located 50 nm distant.
To understand genomic processes such as transcription, translation or splicing, we need to be able to study their spatial and temporal organization at the molecular level. Single-molecule approaches ...provide this opportunity, allowing researchers to monitor molecular conformations, interactions or diffusion quantitatively and in real time in purified systems and in the context of the living cell. This Review introduces the types of application of single-molecule approaches that can enhance our understanding of genome function.
Significance RNA, like DNA, can form double helices held together by the pairing of complementary bases, and such helices are ubiquitous in functional RNAs. Here we apply external forces and torques ...to individual double-stranded RNA molecules to determine the mechanical properties and conformational transitions of these fundamental biological building blocks. For small forces and torques, RNA helices behave like elastic rods, and we have determined their bending, stretching, and twisting stiffness. Surprisingly, we find that RNA shortens when it is overwound, whereas DNA lengthens. Finally, we twist RNA until it buckles and forms a loop, and find the timescale of this transition to be much slower for RNA compared with DNA, suggesting unexpected differences in their flexibilities on short length scales.
RNA plays myriad roles in the transmission and regulation of genetic information that are fundamentally constrained by its mechanical properties, including the elasticity and conformational transitions of the double-stranded (dsRNA) form. Although double-stranded DNA (dsDNA) mechanics have been dissected with exquisite precision, much less is known about dsRNA. Here we present a comprehensive characterization of dsRNA under external forces and torques using magnetic tweezers. We find that dsRNA has a force–torque phase diagram similar to that of dsDNA, including plectoneme formation, melting of the double helix induced by torque, a highly overwound state termed “P-RNA,” and a highly underwound, left-handed state denoted “L-RNA.” Beyond these similarities, our experiments reveal two unexpected behaviors of dsRNA: Unlike dsDNA, dsRNA shortens upon overwinding, and its characteristic transition rate at the plectonemic buckling transition is two orders of magnitude slower than for dsDNA. Our results challenge current models of nucleic acid mechanics, provide a baseline for modeling RNAs in biological contexts, and pave the way for new classes of magnetic tweezers experiments to dissect the role of twist and torque for RNA–protein interactions at the single-molecule level.
DNA wrapping around histone octamers generates nucleosomes, the basic compaction unit of eukaryotic chromatin. Nucleosome stability is carefully tuned to maintain DNA accessibility in transcription, ...replication, and repair. Using freely orbiting magnetic tweezers, which measure the twist and length of single DNA molecules, we monitor the real-time loading of tetramers or complete histone octamers onto DNA by Nucleosome Assembly Protein-1 (NAP1). Remarkably, we find that tetrasomes exhibit spontaneous flipping between a preferentially occupied left-handed state (ΔLk = −0.73) and a right-handed state (ΔLk = +1.0), separated by a free energy difference of 2.3 kBT (1.5 kcal/mol). This flipping occurs without concomitant changes in DNA end-to-end length. The application of weak positive torque converts left-handed tetrasomes into right-handed tetrasomes, whereas nucleosomes display more gradual conformational changes. Our findings reveal unexpected dynamical rearrangements of the nucleosomal structure, suggesting that chromatin can serve as a “twist reservoir,” offering a mechanistic explanation for the regulation of DNA supercoiling in chromatin.
Display omitted
•We monitor assembly of nucleosomes and tetrasomes by NAP1 on DNA in real time•Tetrasomes spontaneously flip between a left- and right-handed conformation•Addition of H2A/H2B to flipping tetrasomes generates stable left-handed nucleosomes•Small positive torques drive tetrasomes from a left-handed into a right-handed state
In eukaryotes, DNA is wrapped in a left-handed fashion around histone protein cores, forming nucleosomes. Vlijm et al. now use real-time monitoring of DNA length and linking number to show that tetrasomes, biologically relevant subnucleosomal structures, exhibit spontaneous flipping between a preferentially occupied left-handed and a right-handed state.
Summary
Background
Maternal fatty acid status during pregnancy might influence foetal immunological development and subsequently the risk of childhood atopic diseases.
Objective
To examine the ...associations of maternal fatty acid levels during pregnancy with airway resistance and inflammation, asthma and eczema, in school‐age children.
Methods
This study among 4976 subjects was embedded in a population‐based prospective cohort study. We measured maternal plasma glycerophospholipid fatty acid levels by gas chromatography during the second trimester of pregnancy (mean gestational age: 20.7 (± 1.1) weeks). At the age of 6 years, airway resistance and inflammation were measured by interrupter technique (Rint) and fractional exhaled nitric oxide (FeNO), and current physician‐diagnosed asthma and eczema were assessed by ISAAC‐based questionnaires. Multiple linear and logistic regression models were adjusted for socio‐demographic, lifestyle and anthropometric factors.
Results
We did not observe consistent associations of maternal total polyunsaturated fatty acid (PUFA), total n‐6 PUFA, total n‐3 PUFA levels and n‐6/n‐3 PUFA ratio during pregnancy with child's Rint and FeNO. Higher maternal total PUFA and total n‐6 PUFA levels were associated with a decreased risk of childhood asthma (odds ratios (95% confidence interval): 0.76 (0.60, 0.97) and 0.71 (0.52, 0.96) per standard deviation score (SDS) increase of total PUFA and total n‐6 PUFA levels, respectively) and with an increased risk of childhood eczema (1.16 (1.05, 1.28) and 1.21 (1.07, 1.37)). The observed associations were partly explained by Linoleic acid (LA, C18:2n‐6) levels. Maternal total n‐3 PUFA levels and n‐6/n‐3 PUFA ratio were not associated with current asthma and eczema. The observed associations were not explained by child's PUFA intake.
Conclusions and Clinical Relevance
Higher maternal total PUFA and total n‐6 PUFA levels during pregnancy seem to influence the risk of atopic diseases in childhood. The underlying mechanisms need to be further explored.
This paper examines the impact of facial cues on leadership emergence. Using evolutionary social psychology, we expand upon implicit and contingent theories of leadership and propose that different ...types of intergroup relations elicit different implicit cognitive leadership prototypes. It is argued that a biologically based hormonal connection between behavior and corresponding facial characteristics interacts with evolutionarily consistent social dynamics to influence leadership emergence. We predict that masculine-looking leaders are selected during intergroup conflict (war) and feminine-looking leaders during intergroup cooperation (peace). Across two experiments we show that a general categorization of leader versus nonleader is an initial implicit requirement for emergence, and at a context-specific level facial cues of masculinity and femininity contingently affect war versus peace leadership emergence in the predicted direction. In addition, we replicate our findings in Experiment 1 across culture using Western and East Asian samples. In Experiment 2, we also show that masculine-feminine facial cues are better predictors of leadership than male-female cues. Collectively, our results indicate a multi-level classification of context-specific leadership based on visual cues imbedded in the human face and challenge traditional distinctions of male and female leadership.
The double-stranded nature of DNA links its replication, transcription and repair to rotational motion and torsional strain. Magnetic tweezers (MT) are a powerful single-molecule technique to apply ...both forces and torques to individual DNA or RNA molecules. However, conventional MT do not track rotational motion directly and constrain the free rotation of the nucleic acid tether. Here we present freely orbiting MT (FOMT) that allow the measurement of equilibrium fluctuations and changes in the twist of tethered nucleic acid molecules. Using a precisely aligned vertically oriented magnetic field, FOMT enable tracking of the rotation angle from straight forward (x,y)-position tracking and permits the application of calibrated stretching forces, without biasing the tether's free rotation. We utilize FOMT to measure the force-dependent torsional stiffness of DNA from equilibrium rotational fluctuations and to follow the assembly of recombination protein A filaments on DNA.
The eukaryotic replicative helicase CMG centrally orchestrates the replisome and leads the way at the front of replication forks. Understanding the motion of CMG on the DNA is therefore key to our ...understanding of DNA replication. In vivo, CMG is assembled and activated through a cell-cycle-regulated mechanism involving 36 polypeptides that has been reconstituted from purified proteins in ensemble biochemical studies. Conversely, single-molecule studies of CMG motion have thus far relied on pre-formed CMG assembled through an unknown mechanism upon overexpression of individual constituents. Here, we report the activation of CMG fully reconstituted from purified yeast proteins and the quantification of its motion at the single-molecule level. We observe that CMG can move on DNA in two ways: by unidirectional translocation and by diffusion. We demonstrate that CMG preferentially exhibits unidirectional translocation in the presence of ATP, whereas it preferentially exhibits diffusive motion in the absence of ATP. We also demonstrate that nucleotide binding halts diffusive CMG independently of DNA melting. Taken together, our findings support a mechanism by which nucleotide binding allows newly assembled CMG to engage with the DNA within its central channel, halting its diffusion and facilitating the initial DNA melting required to initiate DNA replication.
Abstract
Chromatin replication involves the assembly and activity of the replisome within the nucleosomal landscape. At the core of the replisome is the Mcm2-7 complex (MCM), which is loaded onto DNA ...after binding to the Origin Recognition Complex (ORC). In yeast, ORC is a dynamic protein that diffuses rapidly along DNA, unless halted by origin recognition sequences. However, less is known about the dynamics of ORC proteins in the presence of nucleosomes and attendant consequences for MCM loading. To address this, we harnessed an in vitro single-molecule approach to interrogate a chromatinized origin of replication. We find that ORC binds the origin of replication with similar efficiency independently of whether the origin is chromatinized, despite ORC mobility being reduced by the presence of nucleosomes. Recruitment of MCM also proceeds efficiently on a chromatinized origin, but subsequent movement of MCM away from the origin is severely constrained. These findings suggest that chromatinized origins in yeast are essential for the local retention of MCM, which may facilitate subsequent assembly of the replisome.
Over the past few years, it has become increasingly apparent that double-stranded RNA (dsRNA) plays a far greater role in the life cycle of a cell than previously expected. Numerous proteins, ...including helicases, polymerases, and nucleases interact specifically with the double helix of dsRNA. To understand the detailed nature of these dsRNA-protein interactions, the (bio)chemical, electrostatic, and mechanical properties of dsRNA need to be fully characterized. We present measurements of the persistence length of dsRNA using two different single-molecule techniques: magnetic tweezers and atomic force microscopy. We deduce a mean persistence length for long dsRNA molecules of 63.8
±
0.7
nm from force-extension measurements with the magnetic tweezers. We present atomic force microscopy images of dsRNA and demonstrate a new method for analyzing these, which yields an independent, yet consistent value of 62
±
2
nm for the persistence length. The introduction of these single-molecule techniques for dsRNA analysis opens the way for real-time, quantitative analysis of dsRNA-protein interactions.
PDF
