Crucial role for DNA ligase III in mitochondria but not in Xrcc1-dependent repair

Mammalian cells have three ATP-dependent DNA ligases, which are required for DNA replication and repair. Homologues of ligase I (Lig1) and ligase IV (Lig4) are ubiquitous in Eukarya, whereas ligase III (Lig3), which has nuclear and mitochondrial forms, appears to be restricted to vertebrates. Lig3 is implicated in various DNA repair pathways with its partner protein Xrcc1 (ref. 1). Deletion of Lig3 results in early embryonic lethality in mice, as well as apparent cellular lethality, which has precluded definitive characterization of Lig3 function. Here we used pre-emptive complementation to determine the viability requirement for Lig3 in mammalian cells and its requirement in DNA repair. Various forms of Lig3 were introduced stably into mouse embryonic stem (mES) cells containing a conditional allele of Lig3 that could be deleted with Cre recombinase. With this approach, we find that the mitochondrial, but not nuclear, Lig3 is required for cellular viability. Although the catalytic function of Lig3 is required, the zinc finger (ZnF) and BRCA1 carboxy (C)-terminal-related (BRCT) domains of Lig3 are not. Remarkably, the viability requirement for Lig3 can be circumvented by targeting Lig1 to the mitochondria or expressing Chlorella virus DNA ligase, the minimal eukaryal nick-sealing enzyme, or Escherichia coli LigA, an NAD(+)-dependent ligase. Lig3-null cells are not sensitive to several DNA-damaging agents that sensitize Xrcc1-deficient cells. Our results establish a role for Lig3 in mitochondria, but distinguish it from its interacting protein Xrcc1.     

Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However, it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines, along with methylomes of ES cells, somatic cells, and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability, including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation, and differences in CG methylation and histone modifications. Lastly, differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency, providing an iPSC reprogramming signature that is maintained after differentiation.     

Direct measurement of the quantum wavefunction

The wavefunction is the complex distribution used to completely describe a quantum system, and is central to quantum theory. But despite its fundamental role, it is typically introduced as an abstract element of the theory with no explicit definition. Rather, physicists come to a working understanding of the wavefunction through its use to calculate measurement outcome probabilities by way of the Born rule. At present, the wavefunction is determined through tomographic methods, which estimate the wavefunction most consistent with a diverse collection of measurements. The indirectness of these methods compounds the problem of defining the wavefunction. Here we show that the wavefunction can be measured directly by the sequential measurement of two complementary variables of the system. The crux of our method is that the first measurement is performed in a gentle way through weak measurement, so as not to invalidate the second. The result is that the real and imaginary components of the wavefunction appear directly on our measurement apparatus. We give an experimental example by directly measuring the transverse spatial wavefunction of a single photon, a task not previously realized by any method. We show that the concept is universal, being applicable to other degrees of freedom of the photon, such as polarization or frequency, and to other quantum systems--for example, electron spins, SQUIDs (superconducting quantum interference devices) and trapped ions. Consequently, this method gives the wavefunction a straightforward and general definition in terms of a specific set of experimental operations. We expect it to expand the range of quantum systems that can be characterized and to initiate new avenues in fundamental quantum theory.     

Using photoemission spectroscopy to probe a strongly interacting Fermi gas

Ultracold atomic gases provide model systems in which to study many-body quantum physics. Recent experiments using Fermi gases have demonstrated a phase transition to a superfluid state with strong interparticle interactions. This system provides a realization of the 'BCS-BEC crossover' connecting the physics of Bardeen-Cooper-Schrieffer (BCS) superconductivity with that of Bose-Einstein condensates (BECs). Although many aspects of this system have been investigated, it has not yet been possible to measure the single-particle excitation spectrum (a fundamental property directly predicted by many-body theories). Here we use photoemission spectroscopy to directly probe the elementary excitations and energy dispersion in a strongly interacting Fermi gas of (40)K atoms. In the experiments, a radio-frequency photon ejects an atom from the strongly interacting system by means of a spin-flip transition to a weakly interacting state. We measure the occupied density of single-particle states at the cusp of the BCS-BEC crossover and on the BEC side of the crossover, and compare these results to that for a nearly ideal Fermi gas. We show that, near the critical temperature, the single-particle spectral function is dramatically altered in a way that is consistent with a large pairing gap. Our results probe the many-body physics in a way that could be compared to data for the high-transition-temperature superconductors. As in photoemission spectroscopy for electronic materials, our measurement technique for ultracold atomic gases directly probes low-energy excitations and thus can reveal excitation gaps and/or pseudogaps. Furthermore, this technique can provide an analogue of angle-resolved photoemission spectroscopy for probing anisotropic systems, such as atoms in optical lattice potentials.     

Molecular cloning and expression of the major protein kinase C substrate of platelets

In platelets, agonists that stimulate phosphoinositide turnover cause the rapid phosphorylation of a protein of apparent relative molecular mass (Mr) 40-47,000, called P47, by protein kinase C (PKC). Diverse identities have been ascribed to P47 including lipocortin, inositol 1,4,5-trisphosphate 5-phosphomonoesterase, pyruvate dehydrogenase alpha subunit and an actin regulatory protein. We have isolated human P47 clones by immunological screening of a lambda gt11 complementary DNA library from HL-60 cells, a human promyelocytic leukaemia cell line. P47 recombinants thus identified hybridized to a 3.0 kilobase (kb) messenger RNA in mature white blood cell lines; the same mRNA was induced in HL-60 cells during differentiation. A 1,050 base pair (bp) open reading frame that could encode a protein of Mr40,087 was confirmed by comparison with peptide sequences from platelet P47, and by expression of the putative recombinant P47 in E. coli and in vitro. The P47 sequence appears to have been conserved throughout vertebrate evolution, and is not similar to any other known sequence including human lipocortin and the alpha subunit of pyruvate dehydrogenase. The P47 protein contains a potential Ca2+-binding 'EF-hand' structure and a region that strongly resembles known PKC phosphorylation sites.