DNA polymerase lambda (pol ) is a novel family X DNA polymerase that has been suggested to play a role in meiotic recombination and DNA repair. The recent demonstration of an intrinsic 5-deoxyribose-5-phosphate lyase activity in pol supports a function of this enzyme in base excision repair. However, the biochemical properties of the polymerization activity of this enzyme are still largely unknown. We have cloned and purified human pol to homogeneity in a soluble and active form, and we present here a biochemical description of its polymerization features. In support of a role in DNA repair, pol inserts nucleotides in a DNA template-dependent manner and is processive in small gaps containing a 5-phosphate group. These properties, together with its nucleotide insertion fidelity parameters and lack of proofreading activity, indicate that pol is a novel ␤-like DNA polymerase. However, the high affinity of pol for dNTPs (37-fold over pol ␤) is consistent with its possible involvement in DNA transactions occurring under low cellular levels of dNTPs. This suggests that, despite their similarities, pol ␤ and pol have nonredundant in vivo functions.

Spontaneous mutations are the ultimate source of genetic variation and have a prominent role in evolution. RNA viruses such as hepatitis C virus (HCV) have extremely high mutation rates, but these rates have been inferred from a minute fraction of genome sites, limiting our view of how RNA viruses create diversity. Here, by applying high-fidelity ultradeep sequencing to a modified replicon system, we scored >15,000 spontaneous mutations, encompassing more than 90% of the HCV genome. This revealed >1,000-fold differences in mutability across genome sites, with extreme variations even between adjacent nucleotides. We identify base composition, the presence of high- and low-mutation clusters and transition/transversion biases as the main factors driving this heterogeneity. Furthermore, we find that mutability correlates with the ability of HCV to diversify in patients. These data provide a site-wise baseline for interrogating natural selection, genetic load and evolvability in HCV, as well as for evaluating drug resistance and immune evasion risks.

No abstract

Imaging fluorescence resonance energy transfer (FRET) between molecules labeled with fluorescent proteins is emerging as a powerful tool to study changes in ions, ligands, and molecular interactions in their physiological cellular environment. Different methods use either steady-state fluorescence properties or lifetime to quantify the FRET rate. In addition, some provide the absolute FRET efficiency whereas others are simply a relative index very much influenced by the actual settings and instrumentation used, which makes the interpretation of a given FRET rate very difficult. The use and exchange of FRET standards in laboratories using these techniques would help to overcome this drawback. We report here the construction and systematic evaluation of FRET standard probes of varying FRET efficiencies. The standards for intramolecular FRET were protein fusions of the cyan and yellow variants of A. victoria green fluorescent protein (ECFP and citrine) joined by short linkers or larger protein spacers, or ECFP tagged with a tetracysteine motif and labeled with the biarsenical fluorochrome, FlAsH. Negative and positive controls of intermolecular FRET were also used. We compared these FRET standards with up to four FRET quantification methods: ratioing of acceptor to donor emission, donor intensity recovery upon acceptor photobleach, sensitized emission after spectral unmixing of raw images, and fluorescence lifetime imaging (FLIM). The latter was obtained with a frequency-domain setup able to provide high quality lifetime images in less than a second, and is thus very well suited for live cell studies. The FRET rates or indexes of the standards were in good agreement regardless of the method used. For the CFP-tetraCys/FlAsH pair, the rate calculated from CFP quenching was faster than that obtained by FLIM.