Long repeating (TTAGGG) n single-stranded DNA self-condenses into compact beaded filaments stabilized by G-quadruplex formation.

Conformations adopted by long stretches of single-stranded DNA (ssDNA) are of central interest in understanding the architecture of replication forks, R loops, and other structures generated during DNA metabolism in vivo This is particularly so if the ssDNA consists of short nucleotide repeats. Such studies have been hampered by the lack of defined substrates greater than ∼150 nt and the absence of high-resolution biophysical approaches. Here we describe the generation of very long ssDNA consisting of the mammalian telomeric repeat (5'-TTAGGG-3') n , as well as the interrogation of its structure by EM and single-molecule magnetic tweezers (smMT). This repeat is of particular interest because it contains a run of three contiguous guanine residues capable of forming G quartets as ssDNA. Fluorescent-dye exclusion assays confirmed that this G-strand ssDNA forms ubiquitous G-quadruplex folds. EM revealed thick bead-like filaments that condensed the DNA ∼12-fold. The bead-like structures were 5 and 8 nm in diameter and linked by thin filaments. The G-strand ssDNA displayed initial stability to smMT force extension that ultimately released in steps that were multiples ∼28 nm at forces between 6 and 12 pN, well below the >20 pN required to unravel G-quadruplexes. Most smMT steps were consistent with the disruption of the beads seen by EM. Binding by RAD51 distinctively altered the force extension properties of the G-strand ssDNA, suggesting a stochastic G-quadruplex-dependent condensation model that is discussed.

Human Rap1 interacts directly with telomeric DNA and regulates TRF2 localization at the telomere.

The TRF2-Rap1 complex suppresses non-homologous end joining and interacts with DNAPK-C to prevent end joining. We previously demonstrated that hTRF2 is a double strand telomere binding protein that forms t-loops in vitro and recognizes three- and four-way junctions independent of DNA sequence. How the DNA binding characteristics of hTRF2 to DNA is altered in the presence of hRap1 however is not known. Here we utilized EM and quantitative gel retardation to characterize the DNA binding properties of hRap1 and the TRF2-Rap1 complex. Both gel filtration chromatography and mass analysis from two-dimensional projections showed that the TRF2-Rap1 complex exists in solution and binds to DNA as a complex consisting of four monomers each of hRap1 and hTRF2. EM revealed for the first time that hRap1 binds to DNA templates in the absence of hTRF2 with a preference for double strand-single strand junctions in a sequence independent manner. When hTRF2 and hRap1 are in a complex, its affinity for ds telomeric sequences is 2-fold higher than TRF2 alone and more than 10-fold higher for telomeric 3' ends. This suggests that as hTRF2 recruits hRap1 to telomeric sequences, hRap1 alters the affinity of hTRF2 and its binding preference on telomeric DNA. Moreover, the TRF2-Rap1 complex has higher ability to re-model telomeric DNA than either component alone. This finding underlies the importance of complex formation between hRap1 and hTRF2 for telomere function and end protection.

Formation and function of flavin anion radical in cryptochrome 1 blue-light photoreceptor of monarch butterfly.

The monarch butterfly (Danaus plexippus) cryptochrome 1 (DpCry1) belongs in the class of photosensitive insect cryptochromes. Here we purified DpCry1 expressed in a bacterial host and obtained the protein with a stoichiometric amount of the flavin cofactor in the two-electron oxidized, FAD(ox), form. Exposure of the purified protein to light converts the FAD(ox) to the FAD*(-) flavin anion radical by intraprotein electron transfer from a Trp residue in the apoenzyme. To test whether this novel photoreduction reaction is part of the DpCry1 physiological photocycle, we mutated the Trp residue that acts as the ultimate electron donor in flavin photoreduction. The mutation, W328F, blocked the photoreduction entirely but had no measurable effect on the light-induced degradation of DpCry1 in vivo. In light of this finding and the recently published action spectrum of this class of Crys, we conclude that DpCry1 and similar insect cryptochromes do not contain flavin in the FAD(ox) form in vivo and that, most likely, the [see text] photoreduction reaction is not part of the insect cryptochrome photoreaction that results in proteolytic degradation of the photopigment.

Dobutamine stress echocardiography and left ventricular mass changes of mechanical aortic valve replacement in four years follow-up.

AIM: The aim of this study was to investigate the differences in cardiac response to stress according to the size of the prosthetic valve in patients who underwent aortic valve replacement (AVR) and to evaluate the relationship between the size of the prosthetic valve and cardiac recovery-remodeling after the operation. METHODS: Thirty patients who had undergone AVR (12 patients) or double valve replacement (18 patients) underwent dobutamine-stress echocardiography 4.2 years after the operation to evaluate response to stress . They were divided into 2 groups according to valve prosthesis size. The small-size AVR group (group 1, n=17) had prosthetic aortic valves 21 pounds mm; the large-size AVR group (group 2, n=13) had valves >21 mm. Response to stress and preoperative and postoperative echocardiographic findings were compared. Pulsed and continuous-wave Doppler studies were performed at rest and at the end of each stage. Peak and mean aortic gradients, left ventricular diastolic and systolic functions were measured for each group. RESULTS: Dobutamine stress increased heart rate and blood pressure in both groups. Peak pressure gradient across the aortic valve prostheses was 42.1 mm Hg in group 1 and 20.9 mm Hg in group 2 (P<0.05) at rest. After dobutamine infusion, the peak pressure gradient across the aortic valve prostheses increased to 85.1 mm Hg in group 1 and 54 mm Hg in group 2 (P<0.05). Isovolumetric relaxation time returned to normal in both groups following dobutamine infusion; this decrease was significant only in group 1. Patients achieved a decrease in left atrium and left ventricular diameters and volumes, as evidence of remodeling following AVR. Left ventricular mass index (LVMI) decreased from 127.6+/-47.6 to 98.1+/-36.9 and from 159.9+/-16.1 to 125.3+/-10.1 in groups 1 and 2, respectively, but this decline was not statistically significant. CONCLUSIONS: Smaller valves have higher gradients and this significant difference increases under stress. Significant improvement in echocardiographic diameters, cardiac filling volumes and LVMI reflects the benefit of the operation. Cardiac remodeling is independent of valve size, although high transprosthetic gradients occur during stress conditions.