A stretched conformation of DNA with a biological role?

We have discovered a well-defined extended conformation of double-stranded DNA, which we call Σ-DNA, using laser-tweezers force-spectroscopy experiments. At a transition force corresponding to free energy change ΔG = 1·57 ± 0·12 kcal (mol base pair)-1 60 or 122 base-pair long synthetic GC-rich sequences, when pulled by the 3'-3' strands, undergo a sharp transition to the 1·52 ± 0·04 times longer Σ-DNA. Intriguingly, the same degree of extension is also found in DNA complexes with recombinase proteins, such as bacterial RecA and eukaryotic Rad51. Despite vital importance to all biological organisms for survival, genome maintenance and evolution, the recombination reaction is not yet understood at atomic level. We here propose that the structural distortion represented by Σ-DNA, which is thus physically inherent to the nucleic acid, is related to how recombination proteins mediate recognition of sequence homology and execute strand exchange. Our hypothesis is that a homogeneously stretched DNA undergoes a 'disproportionation' into an inhomogeneous Σ-form consisting of triplets of locally B-like perpendicularly stacked bases. This structure may ensure improved fidelity of base-pair recognition and promote rejection in case of mismatch during homologous recombination reaction. Because a triplet is the length of a gene codon, we speculate that the structural physics of nucleic acids may have biased the evolution of recombinase proteins to exploit triplet base stacks and also the genetic code.

Disease-causing point-mutations in metal-binding domains of Wilson disease protein decrease stability and increase structural dynamics.

After cellular uptake, Copper (Cu) ions are transferred from the chaperone Atox1 to the Wilson disease protein (ATP7B) for incorporation into Cu-dependent enzymes in the secretory pathway. Human ATP7B is a large multi-domain membrane-spanning protein which, in contrast to homologues in other organisms, has six similar cytoplasmic metal-binding domains (MBDs). The reason for multiple MBDs is proposed to be indirect modulation of enzymatic activity and it is thus intriguing that point mutations in MBDs can promote Wilson disease. We here investigated, in vitro and in silico, the biophysical consequences of clinically-observed Wilson disease mutations, G85V in MBD1 and G591D in MBD6, incorporated in domain 4. Because G85 and G591 correspond to a conserved Gly found in all MBDs, we introduced the mutations in the well-characterized MBD4. We found the mutations to dramatically reduce the MBD4 thermal stability, shifting the midpoint temperature of unfolding by more than 20 °C. In contrast to wild type MBD4 and MBD4D, MBD4V adopted a misfolded structure with a large ß-sheet content at high temperatures. Molecular dynamic simulations demonstrated that the mutations increased backbone fluctuations that extended throughout the domain. Our findings imply that reduced stability and enhanced dynamics of MBD1 or MBD6 is the origin of ATP7B dysfunction in Wilson disease patients with the G85V or G591D mutation.

Extracellular water and blood pressure in adults with growth hormone (GH) deficiency: a genotype-phenotype association study.

OBJECTIVES: Growth hormone deficiency (GHD) in adults is associated with decreased extracellular water volume (ECW). In response to GH replacement therapy (GHRT), ECW increases and blood pressure (BP) reduces or remains unchanged. Our primary aim was to study the association between polymorphisms in genes related to renal tubular function with ECW and BP before and 1 year after GHRT. The ECW measures using bioimpedance analysis (BIA) and bioimpedance spectroscopy (BIS) were validated against a reference method, the sodium bromide dilution method (Br(-)). DESIGN AND METHODS: Using a candidate gene approach, fifteen single-nucleotide polymorphisms (SNPs) in nine genes with known impact on renal tubular function (AGT, SCNN1A, SCNN1G, SLC12A1, SLC12A3, KCNJ1, STK39, WNK1 and CASR) were genotyped and analyzed for associations with ECW and BP at baseline and with their changes after 1 year of GHRT in 311 adult GHD patients. ECW was measured with the Br(-), BIA, and BIS. RESULTS: Both BIA and BIS measurements demonstrated similar ECW results as the reference method. At baseline, after adjustment for sex and BMI, SNP rs2291340 in the SLC12A1 gene was associated with ECW volume in GHD patients (p = 0.039). None of the SNPs influenced the ECW response to GHRT. One SNP in the SLC12A3 gene (rs11643718; p = 0.024) and three SNPs in the SCNN1G gene [rs5723 (p = 0.02), rs5729 (p = 0.016) and rs13331086 (p = 0.035)] were associated with the inter-individual differences in BP levels at baseline. A polymorphism in the calcium-sensing receptor (CASR) gene (rs1965357) was associated with changes in systolic BP after GHRT (p = 0.036). None of these associations remained statistically significant when corrected for multiple testing. CONCLUSION: The BIA and BIS are as accurate as Br(-) to measure ECW in GHD adults before and during GHRT. Our study provides the first evidence that individual polymorphisms may have clinically relevant effects on ECW and BP in GHD adults.

Force-induced melting of DNA--evidence for peeling and internal melting from force spectra on short synthetic duplex sequences.

Overstretching of DNA occurs at about 60-70 pN when a torsionally unconstrained double-stranded DNA molecule is stretched by its ends. During the transition, the contour length increases by up to 70% without complete strand dissociation. Three mechanisms are thought to be involved: force-induced melting into single-stranded DNA where either one or both strands carry the tension, or a B-to-S transition into a longer, still base-paired conformation. We stretch sequence-designed oligonucleotides in an effort to isolate the three processes, focusing on force-induced melting. By introducing site-specific inter-strand cross-links in one or both ends of a 64 bp AT-rich duplex we could repeatedly follow the two melting processes at 5 mM and 1 M monovalent salt. We find that when one end is sealed the AT-rich sequence undergoes peeling exhibiting hysteresis at low and high salt. When both ends are sealed the AT sequence instead undergoes internal melting. Thirdly, the peeling melting is studied in a composite oligonucleotide where the same AT-rich sequence is concatenated to a GC-rich sequence known to undergo a B-to-S transition rather than melting. The construct then first melts in the AT-rich part followed at higher forces by a B-to-S transition in the GC-part, indicating that DNA overstretching modes are additive.

Tension induces a base-paired overstretched DNA conformation.

Mixed-sequence DNA molecules undergo mechanical overstretching by approximately 70% at 60-70 pN. Since its initial discovery 15 y ago, a debate has arisen as to whether the molecule adopts a new form [Cluzel P, et al. (1996) Science 271:792-794; Smith SB, Cui Y, Bustamante C (1996) Science 271:795-799], or simply denatures under tension [van Mameren J, et al. (2009) Proc Natl Acad Sci USA 106:18231-18236]. Here, we resolve this controversy by using optical tweezers to extend small 60-64 bp single DNA duplex molecules whose base content can be designed at will. We show that when AT content is high (70%), a force-induced denaturation of the DNA helix ensues at 62 pN that is accompanied by an extension of the molecule of approximately 70%. By contrast, GC-rich sequences (60% GC) are found to undergo a reversible overstretching transition into a distinct form that is characterized by a 51% extension and that remains base-paired. For the first time, results proving the existence of a stretched basepaired form of DNA can be presented. The extension observed in the reversible transition coincides with that produced on DNA by binding of bacterial RecA and human Rad51, pointing to its possible relevance in homologous recombination.

Bioelectrical impedance spectroscopy in growth hormone-deficient adults.

This study evaluated water compartment assessment by bioelectrical impedance spectroscopy (BIS) by Xitron 4000B in 164 growth hormone-deficient adults on growth hormone replacement therapy, examined the assumed constant body density and gender-specific resistivities in BIS methodology and evaluated a published BMI-adjusted BIS equation. Body composition was measured by BIS, total body potassium (TBK), tritium dilution and dual-energy x-ray absorptiometry (DXA). Tritium dilution and TBK were combined to a reference method for water compartments. Average difference for total body water (TBW) by tritium dilution and by BIS was 0.6 l in women (p > 0.05) and -0.2 l in men (p > 0.05). Average extracellular water (ECW) by the reference method and by BIS differed 1.5 l in women (p < 0.05) and 1.2 l in men (p < 0.05). Average intracellular water (ICW) by the reference method and by BIS differed -0.9 l in women (p < 0.05) and -1.3 l in men (p < 0.05). However, average ECW and ICW could be successfully estimated by BIS with use of unisex resistivity constants that were derived from this population, although with large individual variation. Average individual body density was lower than assumed. Application of individual body density did not improve agreement between methods. BMI-adjusted equations were not fully accurate in this population.

Body composition in the elderly: reference values and bioelectrical impedance spectroscopy to predict total body skeletal muscle mass.

BACKGROUND & AIMS: To validate the bioelectrical impedance spectroscopy (BIS) model against dual-energy X-ray absorptiometry (DXA), to develop and compare BIS estimates of skeletal muscle mass (SMM) to other prediction equations, and to report BIS reference values of body composition in a population-based sample of 75-year-old Swedes. METHODS: Body composition was measured by BIS in 574 subjects, and by DXA and BIS in a subset of 98 subjects. Data from the latter group was used to develop BIS prediction equations for total body skeletal muscle mass (TBSMM). RESULTS: Average fat free mass (FFM) measured by DXA and BIS was comparable. FFM(BIS) for women and men was 40.6 kg and 55.8 kg, respectively. Average fat free mass index (FFMI) and body fat index (BFI) for women were 15.6 and 11.0. Average FFMI and BFI for men were 18.3 and 8.6. Existing bioelectrical impedance analysis equations to predict SMM were not valid in this cohort. A TBSMM prediction equation developed from this sample had an R(2)(pred) of 0.91, indicating that the equation would explain 91% of the variability in future observations. CONCLUSIONS: BIS correctly estimated average FFM in healthy elderly Swedes. For prediction of TBSMM, a population specific equation was required.