Saccharomyces cerevisiae Dna2 possesses both helicase and endonuclease activities. Its endonuclease activity is essential and well suited to remove RNA-DNA primers of Okazaki fragments. In contrast, its helicase activity, although required for optimal growth, is not essential when the rate of cell growth is reduced. These findings suggest that DNA unwinding activity of Dna2 plays an auxiliary role in Okazaki fragment processing. To address this issue, we examined whether the Dna2 helicase activity influenced its intrinsic endonuclease activity using two mutant proteins, Dna2D657A and Dna2K1080E, which contain only helicase or endonuclease activity, respectively. Experiments performed with a mixture of Dna2D657A and Dna2K1080E enzymes revealed that cleavage of a single-stranded DNA by endonuclease activity of Dna2 occurs while the enzyme translocates along the substrate. In addition, DNA unwinding activity efficiently removed the secondary structure formed in the flap structure, which was further aided by replication protein A. Our results suggest that the Dna2 unwinding activity plays a role in facilitating the removal of the flap DNA by its intrinsic endonuclease activity.

We have previously shown that replication- protein A (RPA), the heterotrimeric single-stranded DNA binding protein of eukaryotes, plays a role in Okazaki fragment processing by acting as a molecular switch between the two endonucleases, Dna2 and Fen1, to ensure the complete removal of primer RNAs in Saccharomyces cerevisiae. The stimulation of Dna2 endonuclease activity by RPA requires direct protein-protein interaction. In this report we have analyzed genetically and biochemically the interaction of Dna2 with RPA. RFA1, the gene encoding the large subunit of RPA, displayed allele-specific interactions with DNA2 that included synthetic lethality and intergenic complementation. In addition, we identified physical and functional interactions between these proteins and found that RPA binds Dna2 predominantly through its large subunit, Rpa1. Consistent with the mapping of synthetic lethal mutations, robust interaction localizes to the C-termini of these proteins. Moreover, the N-terminal domains of Dna2 and Rpa1 appear to be important for a functional interaction because the N-terminal domain of RPA1 was required to maximally stimulate Dna2 endonuclease activity. We propose that a bimodal interaction of Dna2 with Rpa1 is important for Dna2 function both in vivo and in vitro. The relevance of each interaction with respect to the function of the Dna2 endonuclease activity is discussed.

We have identified a novel DNA helicase in humans that belongs to members of the superfamily I helicase and found that it contains a well conserved F-box motif at its N terminus. We have named the enzyme hFBH1 (human F-box DNA helicase 1). Recombinant hFBH1, containing glutathione S-transferase at the N terminus, was expressed in Sf9 cells and purified. In this report, we show that hFBH1 exhibited DNA-dependent ATPase and DNA unwinding activities that displace duplex DNA in the 3 to 5 direction. The hFBH1 enzyme interacted with human SKP1 and formed an SCF (SKP1/Cullin/Fbox) complex together with human Cullin and ROC1. In addition, the SCF complex containing hFBH1 as an Fbox protein displayed ubiquitin ligase activity. We demonstrate that hFBH1 is the first F-box protein that possesses intrinsic enzyme activity. The potential role of the F-box motif and the helicase activity of the enzyme are discussed with regard to regulation of DNA metabolism.

In order to gain insights into the structural basis of the multifunctional Dna2 enzyme involved in Okazaki fragment processing, we performed biochemical, biophysical and genetic studies to dissect the domain structure of Dna2. Proteolytic digestion of Dna2 using subtilisin produced a 127 kDa polypeptide that lacked the 45 kDa N-terminal region of Dna2. Further digestion generated two subtilisin-resistant core fragments of approximately equal size, 58 and 60 kDa. Surprisingly, digestion resulted in a significant (3- to 8-fold) increase in both ATPase and endonuclease activities compared to the intact enzyme. However, cells with a mutant DNA2 allele lacking the corresponding N-terminal region were severely impaired in growth, being unable to grow at 37 degrees C, indicating that the N-terminal region contains a domain critical for a cellular function(s) of Dna2. Analyses of the hydrodynamic properties of and in vivo complex formation by wild-type and/or mutant Dna2 lacking the N-terminal 45 kDa domain revealed that Dna2 is active as the monomer and thus the defect in the mutant Dna2 protein is not due to its inability to multimerize. In addition, we found that the N-terminal 45 kDa domain interacts physically with a central region located between the two catalytic domains. Our results suggest that the N-terminal 45 kDa domain of Dna2 plays a critical role in regulation of the enzymatic activities of Dna2 by serving as a site for intra- and intermolecular interactions essential for optimal function of Dna2 in Okazaki fragment processing. The possible mode of regulation of Dna2 is discussed based upon our recent finding that replication protein A interacts functionally and physically with Dna2 during Okazaki fragment processing.

Previous studies on sulfur cathodes focused on the use of elemental sulfur combined with carbon materials to overcome the issue of low conductivity of sulfur. [12][13][14][15][16][17] As the dissolution of lithium polysulfides in organic electrolytes was revealed to be a main factor obstructing high sulfur utilization, extensive research efforts have focused on the utilization of various porous materials with the physical confinement of sulfur within pores. [12,[18][19][20] While this approach led to significant improvements in the cycle life of lithiumsulfur batteries, the inexpensive production of such materials remains a challenge.This prompted another important strategy of chemical confinements by synthesizing the so-called inverse vulcanized polymers, pioneered by Pyun and co-workers [10,[21][22][23] Sulfur-containing polymers were prepared by simple copolymerization of molten sulfur and molecular dienes, enabling the preparation of cathode active materials in large quantities and at a low cost. The most widely employed linker is 1,3-diisopropenylbenzene (DIB), which showed good thermodynamic compatibility with molten sulfur, thereby offering homogeneous distribution of organosulfur in the cross-linked cathode frameworks with controllable sulfur contents.Lithium-sulfur batteries fabricated using the state-of-the-art sulfur-rich polymers as the cathode active materials have shown an initial discharge capacity of >1200 mAh g −1 and a long-term cycle performance over 500 cycles with a capacity retention of ≈70%. [21][22][23][24] Nevertheless, the conductivity of vulcanized polymers is poor, which is related to the limited rate capability of the resultant lithium-sulfur cells, commonly up to 2C-3C. [21][22][23][24][25][26][27] Without a doubt, the high rate performance is the most pressing challenge facing lithium-sulfur batteries based on organosulfur cathodes. In some recent studies, the successful operation of lithium-sulfur cells at 5C with discharge capacities of ≈630 mAh g −1 was achieved. [28][29][30] This was done by the introduction of conducting polymer linkers in the vulcanization reaction, to increase the electric conductivity of the resultant polymers. [28,31] However, the bulky nature of polymeric linkers results in poor thermodynamic compatibility with sulfur (and lithium polysulfides).Herein, we report a record-high rate performance of sulfur cathodes, i.e., 833 mAh g −1 at 10C, enabled by the synthesis of sulfur-rich polymers comprising functional linkers. Sulfurlinked tetra(allyloxy)-1,4-benzoquinone (S-TABQ) showed an exceptionally low bandgap energy of 2.27 eV, and a Pressing challenges facing future lithium batteries include the realization of a high specific capacity, reliable cycle life, fast charging, and improved safety. Herein, cutting-edge lithium-sulfur batteries based on sulfur-rich polymers that demonstrate a high discharge capacity of 1346 mAh g −1 at 0.1C, a recordhigh rate performance of 833 mAh g −1 at 10C, and long lifetime of 500 cycles with a low capacity decay of 0.052% per ...

It has been suggested that the Schizosaccharomyces pombe Rad50 (Rad50-Rad32-Nbs1) complex is required for the resection of the C-rich strand at telomere ends in taz1-d cells. However, the nuclease-deficient Rad32-D25A mutant can still resect the C-rich strand, suggesting the existence of a nuclease that resects the C-rich strand. Here, we demonstrate that a taz1-d dna2-2C double mutant lost the G-rich overhang at a semipermissive temperature. The amount of G-rich overhang in S phase in the dna2-C2 mutant was lower than that in wild-type cells at the semipermissive temperature. Dna2 bound to telomere DNA in a chromatin immunoprecipitation assay. Moreover, telomere length decreased with each generation after shift of the dna2-2C mutant to the semipermissive temperature. These results suggest that Dna2 is involved in the generation of G-rich overhangs in both wild-type cells and taz1-d cells. The dna2-C2 mutant was not gamma ray sensitive at the semipermissive temperature, suggesting that the ability to process double-strand break (DSB) ends was not affected in the dna2-C2 mutant. Our results reveal that DSB ends and telomere ends are processed by different mechanisms.

Background: Single-and low-copy genes are less likely subject to concerted evolution, thus making themselves ideal tools for studying the origin and evolution of polyploid taxa. Leymus is a polyploid genus with a diverse array of morphology, ecology and distribution in Triticeae. The genomic constitution of Leymus was assigned as NsXm, where Ns was presumed to be originated from Psathyrostachys, while Xm represented a genome of unknown origin. In addition, little is known about the evolutionary history of Leymus. Here, we investigate the phylogenetic relationship, genome donor, and evolutionary history of Leymus based on a single-copy nuclear Acc1 gene.

No abstract