HU is one of the most abundant proteins in bacterial chromosomes and participates in nucleoid compaction and gene regulation. We report experiments using DNA stretching that study the dependence of DNA condensation by HU on force, salt and HU concentration. Previous experiments at sub-physiological salt levels revealed that low concentrations of HU could compact DNA, whereas larger HU concentrations formed a DNA-stiffening complex. Here we report that this bimodal binding behavior depends sensitively on salt concentration. Only the compaction mode was observed for 150 mM and higher NaCl levels, i.e. for physiological salt concentrations. Similar results were obtained for the more physiological salt K-glutamate. Real-time studies of dissociation kinetics revealed that HU unbound slowly (minutes to hours under the conditions studied) but completely for salt concentrations at or above 100 mM NaCl; the lifetime of HU complexes was observed to increase with the HU concentration at which the complexes were formed, and to decrease with salt concentration. Higher salt levels of 300 mM NaCl completely eliminated observable HU binding to DNA. Finally, we observed that the dissociation kinetics depend on force applied to the DNA: increased applied force in the sub-piconewton range accelerates dissociation, suggesting a mechanism for DNA tension to regulate chromosome structure and gene expression.

Mechanics and surface microtopology of the molecular carrier influence cell adhesion, but the mechanisms underlying these effects are not well understood. We used a micropipette adhesion frequency assay to quantify how the carrier stiffness and microtopology affected two-dimensional kinetics of interacting adhesion molecules on two apposing surfaces. Interactions of P-selectin with P-selectin glycoprotein ligand-1 (PSGL-1) were used to demonstrate such effects by presenting the molecules on three carrier systems: human red blood cells (RBCs), human promyelocytic leukemia HL-60 cells, and polystyrene beads. Stiffening the carrier alone or in cooperation with roughing the surface lowered the two-dimensional affinity of interacting molecules by reducing the forward rate but not the reverse rate, whereas softening the carrier and roughing the surface had opposing effects in affecting two-dimensional kinetics. In contrast, the soluble antibody bound with similar three-dimensional affinity to surface-anchored P-selectin or PSGL-1 constructs regardless of carrier stiffness and microtopology. These results demonstrate that the carrier stiffness and microtopology of a receptor influences its rate of encountering and binding a surface ligand but does not subsequently affect the stability of binding. This provides new insights into understanding the rolling and tethering mechanism of leukocytes onto endothelium in both physiological and pathological processes.Selectins are important adhesive molecules that mediate the cell adhesions in such biological processes as platelet thrombosis, inflammatory responses, as well as tumor metastasis (1, 2). For example, selectin-ligand interactions mediate the rolling and tethering of leukocytes onto endothelium in blood flow. Three known selectin members, P-, E-, and L-selectin, have a common structure: an N-terminal, calcium-type lectin (Lec) 5 domain, followed by an epidermal growth factor (EGF)-like module, multiple copies of consensus repeat (CR) units characteristic of complement binding proteins, a transmembrane segment, and a short cytoplasmic domain (3, 4). As a biochemically well-characterized selectin ligand, P-selectin glycoprotein ligand-1 (PSGL-1) consists of homodimer cross-linked by disulfate bonds and binds to selectins through its N-terminal sulfoglycopeptide, which includes three tyrosine sulfates and the core-2 O-glycan (5-8).To mediate cell adhesion, receptors and their ligands must be anchored onto two apposed surfaces, which is the so-called two-dimensional interaction. This is different from three-dimensional binding, where at least one of the receptors and ligands is in the fluid phase. Not only is two-dimensional kinetics of receptor-ligand binding determined by their intrinsic structures, but it also depends on their surface presentation as well as the stiffness and microtopology of molecule-bearing carrier. On the one hand, randomizing the selectin construct or rabbit immunoglobulin G (rIgG) or lowering the length of selectin extending outward from the cell s...

We have studied assembly of chromatin using Xenopus egg extracts and single DNA molecules held at constant tension by using magnetic tweezers. In the absence of ATP, interphase extracts were able to assemble chromatin against DNA tensions of up to 3.5 piconewtons (pN). We observed force-induced disassembly and opening-closing fluctuations, indicating our experiments were in mechanochemical equilibrium. Roughly 50-nm (150-base pair) lengthening events dominated force-driven disassembly, suggesting that the assembled fibers are chiefly composed of nucleosomes. The ATP-depleted reaction was able to do mechanical work of 27 kcal/mol per 50 nm step, which provides an estimate of the free energy difference between core histone octamers on and off DNA. Addition of ATP led to highly dynamic behavior with time courses exhibiting processive runs of assembly and disassembly not observed in the ATP-depleted case. With ATP present, application of forces of 2 pN led to nearly complete fiber disassembly. Our study suggests that ATP hydrolysis plays a major role in nucleosome rearrangement and removal and that chromatin in vivo may be subject to highly dynamic assembly and disassembly processes that are modulated by DNA tension. INTRODUCTIONTranscription, replication, and other in vivo DNA processing in eukaryotes take place in the context of chromatin. The processive nature of these activities, and the necessity to disrupt histone-DNA contacts to accomplish them, suggests that chromatin must be dynamic in its structure, with actively transcribing genes perhaps in a continual state of structural rearrangement. The simplest example of chromatin rearrangement that would allow base pair access is displacement or dissociation of part or all of the histone octamer (Felsenfeld, 1996).Chromosome visualization in vivo gives insight into chromatin dynamics at large length scales (Belmont, 2003;Levi et al., 2005) but is as yet unable to reveal events at the scale of individual nucleosome displacements. A complementary approach is to study individual chromatin fibers by using micromanipulation (Cui and Bustamante, 2000;Ladoux et al., 2000;Bennink et al., 2001;Brower-Toland et al., 2002;Leuba et al., 2003;Claudet et al., 2005;Gemmen et al., 2005;Bancaud et al., 2006). A major objective of such experiments has been the study of mechanically triggered changes in protein-DNA contacts, with an emphasis on force-driven opening of nucleosomes.However, biophysical micromanipulation experiments offer possibilities beyond simply disassembling chromatin by force; experiments in "active" solutions containing chromatin-organizing or chromatin-processing enzymes permit direct observation of chromatin dynamics, and they can reveal details of structure and mechanism concerning compaction of DNA into chromatin, chromatin remodeling, gene expression in chromatin, mitotic chromosome condensation, and how such processes are affected by DNA tension. DNA tension is physiologically relevant because pulling of chromatin is likely to occur in vivo, given the larg...

Barrier-to-autointegration factor (BAF) is a protein that has been proposed to compact retroviral DNA, making it inaccessible as a target for self-destructive integration into itself (autointegration). BAF also plays an important role in nuclear organization. We studied the mechanism of DNA condensation by BAF using total internal reflection fluorescence microscopy. We found that BAF compacts DNA by a looping mechanism. Dissociation of BAF from DNA occurs with multiphasic kinetics; an initial fast phase is followed by a much slower dissociation phase. The mechanistic basis of the broad timescale of dissociation is discussed. This behavior mimics the dissociation of BAF from retroviral DNA within preintegration complexes as monitored by functional assays. Thus the DNA binding properties of BAF may alone be sufficient to account for its association with the preintegration complex.autointegration ͉ BAF (Banf1) ͉ DNA condensation ͉ DNA looping ͉ retrovirus

Determining numbers of proteins bound to large DNAs is important for understanding their chromosomal functions. Protein numbers may be affected by physical factors such as mechanical forces generated in DNA, e.g. by transcription or replication. We performed single-DNA stretching experiments with bacterial nucleoid proteins HU and Fis, verifying that the force–extension measurements were in thermodynamic equilibrium. We, therefore, could use a thermodynamic Maxwell relation to deduce the change of protein number on a single DNA due to varied force. For the binding of both HU and Fis under conditions studied, numbers of bound proteins decreased as force was increased. Our experiments showed that most of the bound HU proteins were driven off the DNA at 6.3 pN for HU concentrations lower than 150 nM; our HU data were fit well by a statistical-mechanical model of protein-induced bending of DNA. In contrast, a significant amount of Fis proteins could not be forced off the DNA at forces up to 12 pN and Fis concentrations up to 20 nM. This thermodynamic approach may be applied to measure changes in numbers of a wide variety of molecules bound to DNA or other polymers. Force-dependent DNA binding by proteins suggests mechano-chemical mechanisms for gene regulation.

Bacterial vaginosis (BV) is a highly prevalent disease in women, and increases the risk of pelvic inflammatory disease. It has been given wide attention because of the high recurrence rate. Traditional diagnostic methods based on microscope providing limited information on the vaginal microbiota increase the difficulty in tracing the development of the disease in bacteria resistance condition. In this study, we used deep-sequencing technology to observe dynamic variation of the vaginal microbiota at three major time points during treatment, at D0 (before treatment), D7 (stop using the antibiotics) and D30 (the 30-day follow-up visit). Sixty-five patients with BV were enrolled (48 were cured and 17 were not cured), and their bacterial composition of the vaginal microbiota was compared. Interestingly, we identified 9 patients might be recurrence. We also introduced a new measurement point of D7, although its microbiota were significantly inhabited by antibiotic and hard to be observed by traditional method. The vaginal microbiota in deep-sequencing-view present a strong correlation to the final outcome. Thus, coupled with detailed individual bioinformatics analysis and deep-sequencing technology, we may illustrate a more accurate map of vaginal microbial to BV patients, which provide a new opportunity to reduce the rate of recurrence of BV.

Histone H1 induces bending and looping of single DNA molecules at nanomolar concentrations. H1 increases the rate of assembly for single chromatin fibers under force in Xenopus egg extracts and protects decondensing sperm nuclei from stretching and fragmenting in egg cytoplasm.

The present study aimed to investigate the prevalence and quantity of Lactobacillus species in the vaginas of healthy Chinese women. Vaginal samples from 92 fertile and 22 postmenopausal healthy Chinese women were analysed using a 16S rRNA gene clone library and species-specific PCR followed by sequencing and real-time PCR. A total of 13 different Lactobacillus species were detected. Species-specific PCR showed that 3 % of the fertile women were colonized by one species of Lactobacillus and 97 % were colonized by two or more species. Among the postmenopausal women, 91 % were colonized by one species of Lactobacillus and 9 % were colonized by two species. In the fertile women, L. iners (82.61 %), L. crispatus (70.65 %) and L. gasseri (67.39 %) were more prevalent than L. jenseniireuteri and L. delbrueckii were not detected, and the other 10 species were detected in just a few samples. The prevalence of these species according to the clone library differed from the prevalence indicated by the species-specific PCR. According to the semiquantitative analysis, the total Lactobacillus DNA concentrations were higher in fertile women than in postmenopausal women. Sixty-one per cent of the fertile women were predominantly colonized by L. iners, 35 % by L. crispatus, and 2 % by L. gasseri. Associations between pairs of Lactobacillus species in fertile women were significant (P,0.05) between the following pairs: L. iners and L. gasseri, L. iners and L. jensenii, L. iners and L. acidophilus, L. gasseri and L. acidophilus, and L. gasseri and L. jensenii. In conclusion, this study provided detailed information on Lactobacillus species colonizing the vaginas of healthy Chinese fertile and postmenopausal women. The study also showed that the diversity of Lactobacillus species in fertile women was higher than in postmenopausal women. According to our study, different techniques, such as species-specific PCR and comparison against a 16S rRNA gene clone library, resulted in different findings regarding species prevalence. These findings highlight the importance of standardization of techniques used for evaluation of bacterial communities. According to our findings regarding species associations, L. iners and L. gasseri may have influences on colonization and proliferation of other vaginal Lactobacillus species.