Clustered Adenine/Thymine Stretches Are Essential for Function of a Fission Yeast Replication Origin

We have determined functional elements required for autonomous replication of the Schizosaccharomyces pombe ars2004 that acts as an intrinsic chromosomal replication origin. Internal deletion analysis of a 940-bp fragment (ars2004M) showed three regions, I to III, to be required for autonomously replicating sequence (ARS) activity. Eight-base-pair substitutions in the 40-bp region I, composed of arrays of adenines on a DNA strand, resulted in a great reduction of ARS activity. Substitutions of region I with synthetic sequences showed that no specific sequence but rather repeats of three or more consecutive adenines or thymines, without interruption by guanine or cytosine, are required for the ARS activity. The 65-bp region III contains 11 repeats of the AAAAT sequence, while the 165-bp region II has short adenine or thymine stretches and a guanine- and cytosine-rich region which enhances ARS activity. All three regions in ars2004M can be replaced with 40-bp poly(dA/dT) fragments without reduction of ARS activity. Although spacer regions in the ars2004M enhance ARS activity, all could be deleted when an 40-bp poly(dA/dT) fragment was added in place of region I. Our results suggest that the origin activity of fission yeast replicators depends on the number of adenine/thymine stretches, the extent of their clustering, and presence of certain replication-enhancing elements.     

Identification of a Binding Region for Human Origin Recognition Complex Proteins 1 and 2 That Coincides with an Origin of DNA Replication

We investigated the binding regions of components of the origin recognition complex (ORC) in the human genome. For this purpose, we performed chromatin immunoprecipitation assays with antibodies against human Orc1 and Orc2 proteins. We identified a binding region for human Orc proteins 1 and 2 in a <1-kbp segment between two divergently transcribed human genes. The region is characterized by CpG tracts and a central sequence rich in AT base pairs. Both, Orc1 and Orc2 proteins are found at the intergenic region in the G(1) phase, but S-phase chromatin contains only Orc2 protein, supporting the notion that Orc1p dissociates from its binding site in the S phase. Sequences corresponding to the intergenic region are highly abundant in a fraction of nascent DNA strands, strongly suggesting that this region not only harbors the binding sites for Orc1 protein and Orc2 protein but also serves as an origin of bidirectional DNA replication.     

Arabidopsis FAB1/PIKfyve Proteins Are Essential for Development of Viable Pollen

Phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂] is a phospholipid that has a role in controlling membrane trafficking events in yeast and animal cells. The function of this lipid in plants is unknown, although its synthesis has been shown to be up-regulated upon osmotic stress in plant cells. PtdIns(3,5)P₂ is synthesized by the PIKfyve/Fab1 family of proteins, with two orthologs, FAB1A and FAB1B, being present in Arabidopsis (Arabidopsis thaliana). In this study, we attempt to address the role of this lipid by analyzing the phenotypes of plants mutated in FAB1A and FAB1B. It was not possible to generate plants homozygous for mutations in both genes, although single mutants were isolated. Both homozygous single mutant plant lines exhibited a leaf curl phenotype that was more marked in FAB1B mutants. Genetic transmission analysis revealed that failure to generate double mutant lines was entirely due to inviability of pollen carrying mutant alleles of both FAB1A and FAB1B. This pollen displayed severe defects in vacuolar reorganization following the first mitotic division of development. The presence of abnormally large vacuoles in pollen at the tricellular stage resulted in the collapse of the majority of grains carrying both mutant alleles. This demonstrates a crucial role for PtdIns(3,5)P₂ in modulating the dynamics of vacuolar rearrangement essential for successful pollen development. Taken together, our results are consistent with PtdIns(3,5)P₂ production being central to cellular responses to changes in osmotic conditions.Phosphoinositides make up a minor fraction of total membrane lipids in all eukaryotic organisms. Their production is spatially restricted to the cytoplasmic leaflet of specific organellar membranes and temporally regulated by phosphatidylinositol (PtdIns) kinases and phosphatases. Three of the five hydoxyl groups of PtdIns can be phosphorylated, either singly or combinatorially, to produce seven different phosphoinositides. These different phosphoinositides can recruit and/or activate proteins with specific phosphoinositide-binding domains and have been implicated in the regulation of many important cellular functions, including membrane trafficking, cell growth, and cytoskeleton remodeling (Di Paolo and De Camilli, 2006).In animal cells, phosphorylation at the 3 position of PtdIns and its phosphorylated derivatives can be carried out by three different classes of PtdIns 3-kinase (classes I–III; Cantley, 2002). Plants and yeast only have class III PtdIns 3-kinases that are orthologs of the Saccharomyces cerevisiae protein Vps34p (Mueller-Roeber and Pical, 2002). Vps34p orthologs are thought to use PtdIns as their sole lipid substrate and produce PtdIns 3-phosphate (PtdIns3P). PtdIns3P is involved in endosomal/lysosomal protein sorting in eukaryotic cells in addition to cellular signaling events (Backer, 2008).In plants, PtdIns3P is essential for normal growth and development. Arabidopsis (Arabidopsis thaliana) plants carrying a VPS34 antisense construct have severe developmental defects (Welters et al., 1994). Furthermore, using pharmacological inhibitors of PtdIns3P production and analysis of transgenic plants defective in downstream signaling from PtdIns3P, it has been shown that this lipid has a role to play in many diverse physiological processes, such as root hair growth (Lee et al., 2008a). The phenotypes observed in studies of PtdIns3P function in plants are consistent with a role in endosomal and vacuolar trafficking in plants (Kim et al., 2001; Lee et al., 2008a), as in other eukaryotes. Recently, an attempt to generate vps34 homozygous mutant plant lines was unsuccessful due to failure of the mutant vps34 allele to transmit through the male germ line (Lee et al., 2008b).Importantly, PtdIns3P is the precursor to another phosphoinositide, PtdIns 3,5-bisphosphate [PtdIns(3,5)P₂], which also has vital roles in endosomal trafficking in eukaryotes (Dove et al., 2009). Thus, it is possible that some of the effects in plants attributed to PtdIns3P in previous studies may actually be due to an inability of cells to produce PtdIns(3,5)P₂. PtdIns(3,5)P₂ is produced by the PtdIns3P 5-kinases PIKfyve and Fab1p in animal and yeast cells, respectively. PIKfyve/Fab1p proteins have an N-terminal FYVE domain necessary for binding to PtdIns3P-containing membranes, a central Cpn60_TCP1 (for HSP chaperonin T complex 1) homology domain, and a C-terminal kinase domain. In Arabidopsis, there are a number of genes encoding putative Fab1p homologs, but only two of them, FAB1A (At4g33240) and FAB1B (At3g14270), encode proteins having FYVE domains at their N termini (Mueller-Roeber and Pical, 2002). It is likely that these proteins are PtdIns3P 5-kinases in Arabidopsis. Despite the importance of PtdIns(3,5)P₂ in yeast and animals, very little is known about its function in plants. However, it has been shown that hyperosmotic stress can induce the rapid synthesis of PtdIns(3,5)P₂ in cell suspension cultures from a number of plant species (Meijer and Munnik, 2003) and in pollen tubes from tobacco (Nicotiana tabacum; Zonia and Munnik, 2004). This production is consistent with a requirement for PtdIns(3,5)P₂ in vacuolar membrane reorganization, as water moves from the vacuole to the cytosol upon cells being placed under hyperosmotic stress. In animal cells, defective PtdIns(3,5)P₂ production leads to cytoplasmic vacuolation of endosome-derived membranes (Ikonomov et al., 2001; Jefferies et al., 2008). It seems that there is a general requirement in all eukaryotes for PtdIns(3,5)P₂ production in endomembrane remodeling. This remodeling could be mediated by proteins that bind to PtdIns(3,5)P₂. A number of candidates have been identified, including yeast Svp1p (Dove et al., 2004), its mammalian homolog WIP149 (Jeffries et al., 2004), CHMP3 (Whitley et al., 2003), and Ent3p (Friant et al., 2003).In this study, we aimed to further investigate the role of PtdIns(3,5)P₂ in plant physiology and the function of PIKfyve/Fab1p orthologs in Arabidopsis by generating mutant plant lines homozygous for T-DNA insertions in both FAB1A and FAB1B. We failed to generate double homozygous fab1a/fab1b knockout plants but observed subtle phenotypes in both fab1a and fab1b single homozygous plants. The data show that pollen with a fab1a/fab1b genotype has an abnormal vacuolar phenotype and does not contribute to the next generation. Our data are consistent with the hypothesis that the male gametophytic defect observed in vps34 mutant pollen (Lee et al., 2008b) is due to an inability of this pollen to generate PtdIns(3,5)P₂ and is not a direct result of the lack of PtdIns3P.     

DNA Hairpin Opening Mediated by the RAG1 and RAG2 Proteins

The lymphoid cell-specific proteins RAG1 and RAG2 initiate V(D)J recombination by cleaving DNA adjacent to recombination signals, generating blunt signal ends and covalently sealed, hairpin coding ends. A critical next step in the reaction is opening of the hairpins, but the factor(s) responsible has not been identified and had been thought to be a ubiquitous component(s) of the DNA repair machinery. Here we demonstrate that RAG1 and RAG2 possess an intrinsic single-stranded nuclease activity capable of nicking hairpin coding ends at or near the hairpin tip. In Mn2+, a synthetic hairpin is nicked 5 nucleotides (nt) 5' of the hairpin tip, with more distant sites of nicking suppressed by HMG2. In Mg2+, hairpins generated by V(D)J cleavage are nicked whereas synthetic hairpins are not. Cleavage-generated hairpins are nicked at the tip and predominantly 1 to 2 nt 5' of the tip. RAG1 and RAG2 may therefore be responsible for initiating the processing of coding ends and for the generation of P nucleotides during V(D)J recombination.     

Identification of a Replication Protein and Repeats Essential for DNA Replication of the Temperate Lactococcal Bacteriophage TP901-1

DNA replication of the temperate lactococcal bacteriophage TP901-1 was shown to involve the gene product encoded by orf13 and the repeats located within the gene. Sequence analysis of 1,500 bp of the early transcribed region of the phage genome revealed a single-stranded DNA binding protein analogue (ORF12) and the putative replication protein (ORF13). The putative origin of replication was identified as series of repeats within orf13 and was shown to confer a TP901-1 resistance phenotype when present in trans. Site-specific mutations were introduced into the replication protein and into the repeats. The mutations were introduced into the TP901-1 prophage by homologous recombination by using a vector with a temperature-sensitive replicon. Subsequent analysis of induced phages showed that the protein encoded by orf13 and the repeats within orf13 were essential for phage TP901-1 amplification. In addition, analyses of internal phage DNA replication showed that the ORF13 protein and the repeats are essential for phage TP901-1 DNA replication in vivo. These results show that orf13 encodes a replication protein and that the repeats within the gene are the origin of replication.     

TbPIF1, a Trypanosoma brucei Mitochondrial DNA Helicase, Is Essential for Kinetoplast Minicircle Replication

Kinetoplast DNA, the trypanosome mitochondrial genome, is a network of interlocked DNA rings including several thousand minicircles and a few dozen maxicircles. Minicircles replicate after release from the network, and their progeny reattach. Remarkably, trypanosomes have six mitochondrial DNA helicases related to yeast PIF1 helicase. Here we report that one of the six, TbPIF1, functions in minicircle replication. RNA interference (RNAi) of TbPIF1 causes a growth defect and kinetoplast DNA loss. Minicircle replication intermediates decrease during RNAi, and there is an accumulation of multiply interlocked, covalently closed minicircle dimers (fraction U). In studying the significance of fraction U, we found that this species also accumulates during RNAi of mitochondrial topoisomerase II. These data indicate that one function of TbPIF1 is an involvement, together with topoisomerase II, in the segregation of minicircle progeny.     

DNA Replication Catalyzed by Herpes Simplex Virus Type 1 Proteins Reveals Trombone Loops at the Fork

Using purified replication factors encoded by herpes simplex virus type 1 and a 70-base minicircle template, we obtained robust DNA synthesis with leading strand products of >20,000 nucleotides and lagging strand fragments from 600 to 9,000 nucleotides as seen by alkaline gel electrophoresis. ICP8 was crucial for the synthesis on both strands. Visualization of the deproteinized products using electron microscopy revealed long, linear dsDNAs, and in 87%, one end, presumably the end with the 70-base circle, was single-stranded. The remaining 13% had multiple single-stranded segments separated by dsDNA segments 500 to 1,000 nucleotides in length located at one end. These features are diagnostic of the trombone mechanism of replication. Indeed, when the products were examined with the replication proteins bound, a dsDNA loop was frequently associated with the replication complex located at one end of the replicated DNA. Furthermore, the frequency of loops correlated with the fraction of DNA undergoing Okazaki fragment synthesis.     

The PS1 Hairpin of Mcm3 Is Essential for Viability and for DNA Unwinding In Vitro

The pre-sensor 1 (PS1) hairpin is found in ring-shaped helicases of the AAA+ family (ATPases associated with a variety of cellular activities) of proteins and is implicated in DNA translocation during DNA unwinding of archaeal mini-chromosome maintenance (MCM) and superfamily 3 viral replicative helicases. To determine whether the PS1 hairpin is required for the function of the eukaryotic replicative helicase, Mcm2-7 (also comprised of AAA+ proteins), we mutated the conserved lysine residue in the putative PS1 hairpin motif in each of the Saccharomyces cerevisiae Mcm2-7 subunits to alanine. Interestingly, only the PS1 hairpin of Mcm3 was essential for viability. While mutation of the PS1 hairpin in the remaining MCM subunits resulted in minimal phenotypes, with the exception of Mcm7 which showed slow growth under all conditions examined, the viable alleles were synthetic lethal with each other. Reconstituted Mcm2-7 containing Mcm3 with the PS1 mutation (Mcm3_K499A) had severely decreased helicase activity. The lack of helicase activity provides a probable explanation for the inviability of the mcm3 _K499A strain. The ATPase activity of Mcm2-7_3K499A was similar to the wild type complex, but its interaction with single-stranded DNA in an electrophoretic mobility shift assay and its associations in cells were subtly altered. Together, these findings indicate that the PS1 hairpins in the Mcm2-7 subunits have important and distinct functions, most evident by the essential nature of the Mcm3 PS1 hairpin in DNA unwinding.     

Origin Binding Protein-Containing Protein-DNA Complex Formation at Herpes Simplex Virus Type 1 oriS: Role in oriS-Dependent DNA Replication

Initiation of herpes simplex virus type 1 (HSV-1) DNA replication during productive infection of fibroblasts and epithelial cells requires attachment of the origin binding protein (OBP), one of seven essential virus-encoded DNA replication proteins, to specific sequences within the two viral origins, oriL and oriS. Whether initiation of DNA replication during reactivation of HSV-1 from neuronal latency also requires OBP is not known. A truncated protein, consisting of the C-terminal 487 amino acids of OBP, termed OBPC, is the product of the HSV UL8.5 gene and binds to origin sequences, although OBPC's role in HSV DNA replication is not yet clear. To characterize protein-DNA complex formation at oriS in cells of neural and nonneural lineage, we used nuclear extracts of HSV-infected nerve growth factor-differentiated PC12 and Vero cells, respectively, as the source of protein in gel shift assays. In both cell types, three complexes (complexes A, B, and C) which contain either OBP or OBPC were shown to bind specifically to a probe which contains the highest-affinity OBP binding site in oriS, site 1. Complex A was shown to contain OBPC exclusively, whereas complexes B and C contained OBP and likely other cellular proteins. By fine-mapping the binding sites of these three complexes, we identified single nucleotides which, when mutated, eliminated formation of all three complexes, or complexes B and C, but not A. In transient DNA replication assays, both mutations significantly impaired oriS-dependent DNA replication, demonstrating that formation of OBP-containing complexes B and C is required for efficient initiation of oriS-dependent DNA replication, whereas formation of the OBPC-containing complex A is insufficient for efficient initiation.     

Ubiquitin Ligase Components Cullin4 and DDB1 Are Essential for DNA Methylation in Neurospora crassa

DNA methylation and H3K9 trimethylation are involved in gene silencing and heterochromatin assembly in mammals and fungi. In the filamentous fungus Neurospora crassa, it has been demonstrated that H3K9 trimethylation catalyzed by histone methyltransferase DIM-5 is essential for DNA methylation. Trimethylated H3K9 is recognized by HP1, which then recruits the DNA methyltransferase DIM-2 to methylate the DNA. Here, we show that in Neurospora, ubiquitin ligase components Cullin4 and DDB1 are essential for DNA methylation. These proteins regulate DNA methylation through their effects on the trimethylation of histone H3K9. In addition, we showed that the E3 ligase activity of the Cul4-based ubiquitin ligase is required for its function in histone H3K9 trimethylation in Neurospora. Furthermore, we demonstrated that Cul4 and DDB1 are associated with the histone methyltransferase DIM-5 protein in vivo. Together, these results suggest a mechanism for DNA methylation control that may be applicable in other eukaryotic organisms.     

Gtsf1l and Gtsf2 Are Specifically Expressed in Gonocytes and Spermatids but Are Not Essential for Spermatogenesis

The unknown protein family 0224 (UPF0224) includes three members that are expressed in germ-line cells in mice: Gtsf1, Gtsf1l, and {"type":"entrez-nucleotide","attrs":{"text":"BC048502","term_id":"28913420"}}BC048502 (Gtsf2). These genes produce proteins with two repeats of the CHHC Zn-finger domain, a predicted RNA-binding motif, in the N terminus. We previously reported that Gtsf1 is essential for spermatogenesis and retrotransposon suppression. In this study, we investigated the expression patterns and functions of Gtsf1l and Gtsf2. Interestingly, Gtsf1l and Gtsf2 were found to be sequentially but not simultaneously expressed in gonocytes and spermatids. Pull-down experiments showed that both GTSF1L and GTSF2 can interact with PIWI-protein complexes. Nevertheless, knocking out Gtsf1, Gtsf2, or both did not cause defects in spermatogenesis or retrotransposon suppression in mice.     

PNT1 Is a C11 Cysteine Peptidase Essential for Replication of the Trypanosome Kinetoplast

The structure of a C11 peptidase PmC11 from the gut bacterium, Parabacteroides merdae, has recently been determined, enabling the identification and characterization of a C11 orthologue, PNT1, in the parasitic protozoon Trypanosoma brucei. A phylogenetic analysis identified PmC11 orthologues in bacteria, archaea, Chromerids, Coccidia, and Kinetoplastida, the latter being the most divergent. A primary sequence alignment of PNT1 with clostripain and PmC11 revealed the position of the characteristic His-Cys catalytic dyad (His⁹⁹ and Cys¹³⁶), and an Asp (Asp¹³⁴) in the potential S₁ binding site. Immunofluorescence and cryoelectron microscopy revealed that PNT1 localizes to the kinetoplast, an organelle containing the mitochondrial genome of the parasite (kDNA), with an accumulation of the protein at or near the antipodal sites. Depletion of PNT1 by RNAi in the T. brucei bloodstream form was lethal both in in vitro culture and in vivo in mice and the induced population accumulated cells lacking a kinetoplast. In contrast, overexpression of PNT1 led to cells having mislocated kinetoplasts. RNAi depletion of PNT1 in a kDNA independent cell line resulted in kinetoplast loss but was viable, indicating that PNT1 is required exclusively for kinetoplast maintenance. Expression of a recoded wild-type PNT1 allele, but not of an active site mutant restored parasite viability after induction in vitro and in vivo confirming that the peptidase activity of PNT1 is essential for parasite survival. These data provide evidence that PNT1 is a cysteine peptidase that is required exclusively for maintenance of the trypanosome kinetoplast.     

None of the Rotor Residues of F1-ATPase Are Essential for Torque Generation

F₁-ATPase is a powerful rotary molecular motor that can rotate an object several hundred times as large as the motor itself against the viscous friction of water. Forced reverse rotation has been shown to lead to ATP synthesis, implying that the mechanical work against the motor’s high torque can be converted into the chemical energy of ATP. The minimal composition of the motor protein is α₃β₃γ subunits, where the central rotor subunit γ turns inside a stator cylinder made of alternately arranged α₃β₃ subunits using the energy derived from ATP hydrolysis. The rotor consists of an axle, a coiled coil of the amino- and carboxyl-terminal α-helices of γ, which deeply penetrates the stator cylinder, and a globular protrusion that juts out from the stator. Previous work has shown that, for a thermophilic F₁, significant portions of the axle can be truncated and the motor still rotates a submicron sized bead duplex, indicating generation of up to half the wild-type (WT) torque. Here, we inquire if any specific interactions between the stator and the rest of the rotor are needed for the generation of a sizable torque. We truncated the protruding portion of the rotor and replaced part of the remaining axle residues such that every residue of the rotor has been deleted or replaced in this or previous truncation mutants. This protrusionless construct showed an unloaded rotary speed about a quarter of the WT, and generated one-third to one-half of the WT torque. No residue-specific interactions are needed for this much performance. F₁ is so designed that the basic rotor-stator interactions for torque generation and control of catalysis rely solely upon the shape and size of the rotor at very low resolution. Additional tailored interactions augment the torque to allow ATP synthesis under physiological conditions.