Details of ssDNA annealing revealed by an HSV-1 ICP8-ssDNA binary complex

Infected cell protein 8 (ICP8) from herpes simplex virus 1 was first identified as a single-strand (ss) DNA-binding protein. It is essential for, and abundant during, viral replication. Studies in vitro have shown that ICP8 stimulates model replication reactions, catalyzes annealing of complementary ssDNAs and, in combination with UL12 exonuclease, will catalyze ssDNA annealing homologous recombination. DNA annealing and strand transfer occurs within large oligomeric filaments of ssDNAbound ICP8. We present the first 3D reconstruction of a novel ICP8-ssDNA complex, which seems to be the basic unit of the DNA annealing machine. The reconstructed volume consists of two nonameric rings containing ssDNA stacked on top of each other, corresponding to a molecular weight of 2.3 MDa. Fitting of the ICP8 crystal structure suggests a mechanism for the annealing reaction catalyzed by ICP8, which is most likely a general mechanism for protein-driven DNA annealing

Details of ssDNA annealing revealed by an HSV-1 ICP8-ssDNA binary complex

Infected cell protein 8 (ICP8) from herpes simplex virus 1 was first identified as a single-strand (ss) DNA-binding protein. It is essential for, and abundant during, viral replication. Studies in vitro have shown that ICP8 stimulates model replication reactions, catalyzes annealing of complementary ssDNAs and, in combination with UL12 exonuclease, will catalyze ssDNA annealing homologous recombination. DNA annealing and strand transfer occurs within large oligomeric filaments of ssDNAbound ICP8. We present the first 3D reconstruction of a novel ICP8-ssDNA complex, which seems to be the basic unit of the DNA annealing machine. The reconstructed volume consists of two nonameric rings containing ssDNA stacked on top of each other, corresponding to a molecular weight of 2.3 MDa. Fitting of the ICP8 crystal structure suggests a mechanism for the annealing reaction catalyzed by ICP8, which is most likely a general mechanism for protein-driven DNA annealing

Details of ssDNA annealing revealed by an HSV-1 ICP8–ssDNA binary complex

Infected cell protein 8 (ICP8) from herpes simplex virus 1 was first identified as a single-strand (ss) DNA-binding protein. It is essential for, and abundant during, viral replication. Studies in vitro have shown that ICP8 stimulates model replication reactions, catalyzes annealing of complementary ssDNAs and, in combination with UL12 exonuclease, will catalyze ssDNA annealing homologous recombination. DNA annealing and strand transfer occurs within large oligomeric filaments of ssDNA-bound ICP8. We present the first 3D reconstruction of a novel ICP8–ssDNA complex, which seems to be the basic unit of the DNA annealing machine. The reconstructed volume consists of two nonameric rings containing ssDNA stacked on top of each other, corresponding to a molecular weight of 2.3 MDa. Fitting of the ICP8 crystal structure suggests a mechanism for the annealing reaction catalyzed by ICP8, which is most likely a general mechanism for protein-driven DNA annealing

Concerted Loading of Mcm2–7 Double Hexamers around DNA during DNA Replication Origin Licensing

The licensing of eukaryotic DNA replication origins, which ensures once per cell cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs). Mcm2-7 forms the core of the replicative DNA helicase, which is inactive in the pre-RC. The ATP-dependent Mcm2-7 loading reaction requires the Origin Recognition Complex (ORC), Cdc6 and Cdt1. We have reconstituted Mcm2-7 loading with purified budding yeast proteins. Using biochemical approaches and electron microscopy, we show that single heptamers of Cdt1·Mcm2-7 are loaded cooperatively into stable, head-to-head Mcm2-7 double hexamers connected via N-terminal rings. DNA runs through a central channel in the double hexamer, and, once loaded, Mcm2-7 can slide passively along double-stranded DNA. Our work has significant implications for understanding how eukaryotic DNA replication origins are chosen and licensed, how replisomes assemble during initiation and how unwinding occurs during DNA replication

The Killing of African Trypanosomes by Ethidium Bromide

Introduced in the 1950s, ethidium bromide (EB) is still used as an anti-trypanosomal drug for African cattle although its mechanism of killing has been unclear and controversial. EB has long been known to cause loss of the mitochondrial genome, named kinetoplast DNA (kDNA), a giant network of interlocked minicircles and maxicircles. However, the existence of viable parasites lacking kDNA (dyskinetoplastic) led many to think that kDNA loss could not be the mechanism of killing. When recent studies indicated that kDNA is indeed essential in bloodstream trypanosomes and that dyskinetoplastic cells survive only if they have a compensating mutation in the nuclear genome, we investigated the effect of EB on kDNA and its replication. We here report some remarkable effects of EB. Using EM and other techniques, we found that binding of EB to network minicircles is low, probably because of their association with proteins that prevent helix unwinding. In contrast, covalently-closed minicircles that had been released from the network for replication bind EB extensively, causing them, after isolation, to become highly supertwisted and to develop regions of left-handed Z-DNA (without EB, these circles are fully relaxed). In vivo, EB causes helix distortion of free minicircles, preventing replication initiation and resulting in kDNA loss and cell death. Unexpectedly, EB also kills dyskinetoplastic trypanosomes, lacking kDNA, by inhibiting nuclear replication. Since the effect on kDNA occurs at a >10-fold lower EB concentration than that on nuclear DNA, we conclude that minicircle replication initiation is likely EB's most vulnerable target, but the effect on nuclear replication may also contribute to cell killing

The NAD+-dependent protein deacetylase activity of SIRT1 is regulated by its oligomeric status

SIRT1, a NAD+-dependent protein deacetylase, is an important regulator in cellular stress response and energy metabolism. While the list of SIRT1 substrates is growing, how the activity of SIRT1 is regulated remains unclear. We have previously reported that SIRT1 is activated by phosphorylation at a conserved Thr522 residue in response to environmental stress. Here we demonstrate that phosphorylation of Thr522 activates SIRT1 through modulation of its oligomeric status. We provide evidence that nonphosphorylated SIRT1 protein is aggregation-prone in vitro and in cultured cells. Conversely, phosphorylated SIRT1 protein is largely in the monomeric state and more active. Our findings reveal a novel mechanism for environmental regulation of SIRT1 activity, which may have important implications in understanding the molecular mechanism of stress response, cell survival, and aging

A real-time DNase assay (ReDA) based on PicoGreen® fluorescence

DNA nucleases (DNases) perform a wide variety of important cellular functions and are also very useful for research and in biotechnological applications. Due to the biological and technological importance of DNases and their use in a wide range of applications, DNase activity assays are essential. Traditional DNase assays employ radiolabeled DNA substrates and require separation of the products of the reaction from the unreacted substrate before quantification of enzyme activity. As a consequence, these methods are discontinuous. In this report, we describe a continuous DNase assay based on the differential fluorescence output of a DNA dye ligand called PicoGreen®. The assay was developed to characterize a processive dsDNA exonuclease, lambda exonuclease. The assay appears to have general utility as it is also suitable for measuring the DNA digestion activities of a processive helicase/nuclease, RecBCD, a distributive exonuclease, T7 gene 6 exonuclease, and an endonuclease, DNaseI. The benefits of, and limitations to, the method are discussed

Schizosacchromyces pombe Dpb2 Binds to Origin DNA Early in S Phase and Is Required for Chromosomal DNA Replication

Genetic evidence suggests that DNA polymerase epsilon (Pol ε) has a noncatalytic essential role during the early stages of DNA replication initiation. Herein, we report the cloning and characterization of the second largest subunit of Pol ε in fission yeast, called Dpb2. We demonstrate that Dpb2 is essential for cell viability and that a temperature-sensitive mutant of dpb2 arrests with a 1C DNA content, suggesting that Dpb2 is required for initiation of DNA replication. Using a chromatin immunoprecipitation assay, we show that Dpb2, binds preferentially to origin DNA at the beginning of S phase. We also show that the C terminus of Pol ε associates with origin DNA at the same time as Dpb2. We conclude that Dpb2 is an essential protein required for an early step in DNA replication. We propose that the primary function of Dpb2 is to facilitate assembly of the replicative complex at the start of S phase. These conclusions are based on the novel cell cycle arrest phenotype of the dpb2 mutant, on the previously uncharacterized binding of Dpb2 to replication origins, and on the observation that the essential function of Pol ε is not dependent on its DNA synthesis activity