Processing of DNA Polymerase-Blocking Lesions during Genome Replication Is Spatially and Temporally Segregated from Replication Forks.

Tracing DNA repair factors by fluorescence microscopy provides valuable information about how DNA damage processing is orchestrated within cells. Most repair pathways involve single-stranded DNA (ssDNA), making replication protein A (RPA) a hallmark of DNA damage and replication stress. RPA foci emerging during S phase in response to tolerable loads of polymerase-blocking lesions are generally thought to indicate stalled replication intermediates. We now report that in budding yeast they predominantly form far away from sites of ongoing replication, and they do not overlap with any of the repair centers associated with collapsed replication forks or double-strand breaks. Instead, they represent sites of postreplicative DNA damage bypass involving translesion synthesis and homologous recombination. We propose that most RPA and recombination foci induced by polymerase-blocking lesions in the replication template are clusters of repair tracts arising from replication centers by polymerase re-priming and subsequent expansion of daughter-strand gaps over the course of S phase.

pH-Sensitive N,N-Dimethylalkane-1-amine N-Oxides in DNA Delivery: From Structure to Transfection Efficiency.

pH-sensitive liposomes composed of homologues of series of N,N-dimethylalkane-1-amine N-oxides (CnNO, n = 8-18, where n is the number of carbon atoms in the alkyl substituent) and neutral phospholipid dioleoylphosphatidylethanolamine (DOPE) were prepared at two molar ratios (CnNO/DOPE = 0.4:1 and 1:1) and tested for their in vitro transfection activity. Several techniques (SAXS/WAXS, UV-vis, zeta potential measurements, confocal microscopy) were applied to characterize the system in an effort to unravel the relationship among the transfection efficiency, structure, and composition of the lipoplexes. The transfection efficiency of CnNO/DOPE for plasmid DNA in U2OS cells follows a quasi-parabolic dependence on CnNO's alkyl substituent length with a maximum at n = 16. The transfection efficiency of CnNO/DOPE (n = 12-18) lipoplexes was found to be higher than that of commercially available Lipofectamine 2000. C16NO/DOPE also positively transfected HEK 293T and HeLa cells. Small-angle X-ray scattering (SAXS) shows large structural diversity depending on the complex's composition and pH. Transfection efficiencies mediated by two structures, either a condensed lamellar (Lαc) or epitaxially connected Lαc and a condensed inverted hexagonal (HIIc) phase (Lαc & HIIc), were found to be very similar. The change in pH from acidic to neutral induces phase transition Lαc & HIIc → QII + Lα, with cubic phase QII of the Pn3m space group. QII detected in lipoplexes of most efficient composition CnNO/DOPE (n = 16 and 18) facilitates DNA release and promotes its internalization in the cell.

DNA-DOPE-gemini surfactants complexes at low surface charge density: from structure to transfection efficiency.

DNA condensation, structure and transfection efficiency of complexes formed by gemini surfactants alkane-α,ω-diyl-bis(dodecyldimethylammonium bromide)s (CnGS12, n = 3, 6 and 12 is the number of alkane spacer carbons), dioleoylphosphatidylethanolamine (CnGS12/DOPE = 0.3 mol/mol) and DNA at low surface charge density were investigated through different techniques. Small angle X-ray diffraction showed a condensed lamellar phase with marked dependence of DNA-DNA distance on (+/-) charge ratio. High ionic strength of hydrating medium screens the interaction DNA - CnGS12/DOPE and complexed DNA represented maximally ~ 45-60% of total DNA in the solution as derived from fluorescence and UV-VIS spectroscopy. The in vitro transfection efficiency of CnGS12/DOPE liposomes on mammalian HEK 293 cell line was spacer length-dependent. C12GS12/DOPE/DNA complexes exhibited the best transfection efficiency (~ 18% GFP-expressing cells relative to all viable cells) accompanied by ~ 89% cell viability.

Water-Soluble NIR-Absorbing Rylene Chromophores for Selective Staining of Cellular Organelles.

Biocompatible organic dyes emitting in the near-infrared are highly desirable in fluorescence imaging techniques. Herein we report a synthetic approach for building novel small peri-guanidine-fused naphthalene monoimide and perylene monoimide chromophores. The presented structures possess near-infrared absorption and emission, high photostability, and good water solubility. After a fast cellular uptake, they selectively stain mitochondria with a low background in live and fixed cells. They can be additionally modified in a one-step reaction with functional groups for covalent labeling of proteins. The low cytotoxicity allows a long time exposure of live cells to the dyes without the necessity of washing. Successful application in localization super-resolution microscopy was demonstrated in phosphate-buffered saline without any reducing or oxidizing additives.

Membrane-mediated interactions--a physico-chemical basis for protein sorting.

Sorting of membrane proteins in eukaryotic cells is a complex yet vital task that involves several 10,000 molecular players. Sorting takes place not only along the early secretory pathway, i.e., between the endoplasmic reticulum and the Golgi apparatus, but also between other organelles, including exchange with the cell's plasma membrane. Traditionally, specific binary interactions between proteins have been made responsible for most of the protein sorting. A more active role of lipids, however, became visible in recent years. Not only do lipids in complex membranes show domain formation that may support/suppress sorting events, but also collective, membrane-mediated interactions have emerged as a robust physico-chemical mechanism to drive protein sorting. Here, we will review recent insights into these aspects.

Protein sorting and membrane-mediated interactions.

Sorting of membrane proteins is of vital importance for living cells. Indeed, roughly one-third of a eukaryotic cell's proteome consists of peripheral and transmembrane proteins. These need to be properly distributed and dynamically maintained at distinct locations in the compartmentalized cell, and one may wonder how proteins determine where, when, and how to travel to reach a specific organelle. While specific binary interactions between proteins have been invoked in explaining the trafficking and sorting processes, a more active role of lipids in this context has become visible in recent years. In particular, membrane-mediated interactions have been suggested to serve as a robust physicochemical mechanism to facilitate protein sorting. Here, we will review some recent insights into these aspects.

The structural diversity of DNA-neutral phospholipids-divalent metal cations aggregates: a small-angle synchrotron X-ray diffraction study.

We investigate the structure of aggregates formed due to DNA interaction with saturated neutral phosphatidylcholines [dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine] in presence of Ca(2+) and Mg(2+) cations using simultaneous synchrotron small- and wide-angle X-ray diffractions. For DPPC:DNA = 3:1 mol/base and in the range of 1-50 mM Ca(2+), the diffractograms show structural heterogeneity of aggregates. We observe the coexistence of two lamellar phases in aggregates prepared at 1 mM Ca(2+): L(x) phase with the DNA strands (of unknown organization) intercalated in water layers between adjacent lipid bilayers and L(DPPC) phase of DPPC bilayers without any divalent cations and DNA strands. Aggregates prepared in the range 2-50 mM Ca(2+) show a condensed gel lamellar phase L (g) (c) with the lipid bilayer periodicity d approximately 8.0 nm, and the DNA-DNA interhelical distance d (DNA) approximately 5.1 nm. The increase of temperature induces the decrease in the intensity and the increase in the width of the DNA related peak. In the fluid state, the condensed lamellar phase L (alpha) (c) gradually converts into L(x) phase. The aggregates do not exhibit rippled P(beta) phase. The thermal behaviour of aggregates was investigated in the range 20-80 degrees C. Applying heating-cooling cycles, the aggregates converted into energetically more favourable structure: a condensed lamellar phase L(c) (or L(x)) is preserved or we observe lateral segregation of the DNA strands and metal cations (L(x) phase) in coexistence with L(PC) phase of pure phospholipids.

The structure of DNA-DOPC aggregates formed in presence of calcium and magnesium ions: a small-angle synchrotron X-ray diffraction study.

The structure of aggregates formed due to DNA interaction with dioleoylphosphatidylcholine (DOPC) vesicles in presence of Ca(2+) and Mg(2+) cations was investigated using synchrotron small-angle X-ray diffraction. For DOPC/DNA=1:1 mol/base and in the range of concentration of the cation(2+) 0-76.5 mM, the diffractograms show the coexistence of two lamellar phases: L(x) phase with repeat distance d(Lx) approximately 8.26-7.39 nm identified as a phase where the DNA strands are intercalated in water layers between adjacent lipid bilayers, and L(DOPC) phase with repeat distance d(DOPC) approximately 6.45-5.65 nm identified as a phase of partially dehydrated DOPC bilayers without any divalent cations and DNA strands. The coexistence of these phases was investigated as a function of DOPC/DNA molar ratio, length of DNA fragments and temperature. If the amount of lipid increases, the fraction of partially dehydrated L(DOPC) phase is limited, depends on the portion of DNA in the sample and also on the length of DNA fragments. Thermal behaviour of DOPC+DNA+Ca(2+) aggregates was investigated in the range 20-80 degrees C. The transversal thermal expansivities of both phases were evaluated.

The structure of DNA-DLPC-cationic gemini surfactant aggregates: a small angle synchrotron X-ray diffraction study.

The structure of aggregates formed by interaction of DNA with unilamellar dilauroylphosphatidylcholine (DLPC) vesicles (DNA:DLPC=1:1 base/mol) in the presence of gemini surfactant butane-1,4-diyl-bis(dodecyldimethylammonium bromide) (C12GS) was investigated using synchrotron small angle X-ray diffraction. In the concentration range C12GS+:DLPC< or =1 mol/mol, a condensed lamellar Lalphac phase was found with a repeat period of lipid bilayer stacking in the range d approximately 5.70-6.53 nm and the DNA interhelical distance d(DNA) approximately 3.52-3.99 nm, depending on the concentration of C12GS. At molar ratio C12GS+:DLPC> or =0.35:1, the diffractograms have shown the presence of a second lamellar phase with the repeat period d approximately 5.31 nm which slightly decreases with increasing concentration of C12GS+. The increasing fraction of this phase in the aggregates with increasing concentration of C12GS supports the association of this phase with microscopic domains enriched by surfactant molecules. The temperature behaviour of aggregates was investigated in the range 25-60 degrees C and the transversal thermal expansivities of the observed phases were determined.

Bilayer thickness in unilamellar extruded 1,2-dimyristoleoyl and 1,2-dierucoyl phosphatidylcholine vesicles: SANS contrast variation study of cholesterol effect.

Small-angle neutron scattering on extruded unilamellar vesicles in water was used to study bilayer thickness when cholesterol (CHOL) was added at 44.4 mol% to 1,2-dimyristoleoylphosphatidylcholine (diC14:1PC) and 1,2-dierucoylphosphatidylcholine (diC22:1PC) bilayers. Using the (1)H(2)O/(2)H(2)O contrast variation and the small-angle form of Kratky-Porod approximation, the bilayer gyration radii at infinite contrast R(g,infinity) and the bilayer thickness parameters d(g,infinity) = 12(0.5)R(g,infinity) were obtained at 30 degrees C. Addition of cholesterol to diC14:1PC increased the d(g,infinity) from 3.72 +/- 0.02 to 4.26 +/- 0.01 nm, while in the diC22:1PC bilayers the d(g,infinity) change observed was within the experimental error: +0.23 +/- 0.23 nm.