Genomic and genetic analyses of diversity and plant interactions of Pseudomonas fluorescens

Background: Pseudomonas fluorescens are common soil bacteria that can improve plant health through nutrient cycling, pathogen antagonism and induction of plant defenses. The genome sequences of strains SBW25 and Pf0-1 were determined and compared to each other and with P. fluorescens Pf-5. A functional genomic in vivo expression technology (IVET) screen provided insight into genes used by P. fluorescens in its natural environment and an improved understanding of the ecological significance of diversity within this species. Results: Comparisons of three P. fluorescens genomes (SBW25, Pf0-1, Pf-5) revealed considerable divergence: 61% of genes are shared, the majority located near the replication origin. Phylogenetic and average amino acid identity analyses showed a low overall relationship. A functional screen of SBW25 defined 125 plant-induced genes including a range of functions specific to the plant environment. Orthologues of 83 of these exist in Pf0-1 and Pf-5, with 73 shared by both strains. The P. fluorescens genomes carry numerous complex repetitive DNA sequences, some resembling Miniature Inverted-repeat Transposable Elements (MITEs). In SBW25, repeat density and distribution revealed 'repeat deserts' lacking repeats, covering approximately 40% of the genome. Conclusions: P. fluorescens genomes are highly diverse. Strain-specific regions around the replication terminus suggest genome compartmentalization. The genomic heterogeneity among the three strains is reminiscent of a species complex rather than a single species. That 42% of plant-inducible genes were not shared by all strains reinforces this conclusion and shows that ecological success requires specialized and core functions. The diversity also indicates the significant size of genetic information within the Pseudomonas pan genome.

Trypanosome Prereplication Machinery Contains a Single Functional Orc1/Cdc6 Protein, Which Is Typical of Archaea

In unicellular eukaryotes, such as Saccharomyces cerevisiae, and in multicellular organisms, the replication origin is recognized by the heterohexamer origin recognition complex (ORC) containing six proteins, Orc1 to Orc6, while in members of the domain Archaea, the replication origin is recognized by just one protein, Orc1/Cdc6; the sequence of Orc1/Cdc6 is highly related to those of Orc1 and Cdc6. Similar to Archaea, trypanosomatid genomes contain only one gene encoding a protein named Orc1. Since trypanosome Orc1 is also homologous to Cdc6, in this study we named the Orc1 protein from trypanosomes Orc1/Cdc6. Here we show that the recombinant Orc1/Cdc6 from Trypanosoma cruzi (TcOrc1/Cdc6) and from Trypanosoma brucei (TbOrc1/Cdc6) present ATPase activity, typical of prereplication machinery components. Also, TcOrc1/Cdc6 and TbOrc1/Cdc6 replaced yeast Cdc6 but not Orc1 in a phenotypic complementation assay. The induction of Orc1/Cdc6 silencing by RNA interference in T. brucei resulted in enucleated cells, strongly suggesting the involvement of Orc1/Cdc6 in DNA replication. Orc1/Cdc6 is expressed during the entire cell cycle in the nuclei of trypanosomes, remaining associated with chromatin in all stages of the cell cycle. These results allowed us to conclude that Orc1/Cdc6 is indeed a member of the trypanosome prereplication machinery and point out that trypanosomes carry a prereplication machinery that is less complex than other eukaryotes and closer to archaea.

RefZ Facilitates the Switch from Medial to Polar Division during Spore Formation in Bacillus subtilis

During sporulation, Bacillus subtilis redeploys the division protein FtsZ from midcell to the cell poles, ultimately generating an asymmetric septum. Here, we describe a sporulation-induced protein, RefZ, that facilitates the switch from a medial to a polar FtsZ ring placement. The artificial expression of RefZ during vegetative growth converts FtsZ rings into FtsZ spirals, arcs, and foci, leading to filamentation and lysis. Mutations in FtsZ specifically suppress RefZ-dependent division inhibition, suggesting that RefZ may target FtsZ. During sporulation, cells lacking RefZ are delayed in polar FtsZ ring formation, spending more time in the medial and transition stages of FtsZ ring assembly. A RefZ-green fluorescent protein (GFP) fusion localizes in weak polar foci at the onset of sporulation and as a brighter midcell focus at the time of polar division. RefZ has a TetR DNA binding motif, and point mutations in the putative recognition helix disrupt focus formation and abrogate cell division inhibition. Finally, chromatin immunoprecipitation assays identified sites of RefZ enrichment in the origin region and near the terminus. Collectively, these data support a model in which RefZ helps promote the switch from medial to polar division and is guided by the organization of the chromosome. Models in which RefZ acts as an activator of FtsZ ring assembly near the cell poles or as an inhibitor of the transient medial ring at midcell are discussed.

Native bradyrhizobial symbionts of Lupinus mariae-josephae, a unique endemism thriving in alkaline soils in Eastern Spain

Lupinus mariae-josephae (Lmj) es una especie de lupino endémica de una pequeña y específica área de Comunidad Valenciana (Este de España), donde prospera en suelos alcalinoscalcáreos, un hábitat singular para los altramuces, que crecen preferentemente en suelos ácidos o neutros. Esto hace de Lmj una especie de lupino única. Cuando se inició este trabajo, la extensión conocida de este endemismo abarcaba unos 700 kilómetros cuadrados, confinados en la provincia de Valencia. En esta área, Lmj prospera en pequeñas poblaciones aisladas que contienen un número reducido de plantas por lo que se la consideró una especie en peligro de extinción. Todos los esfuerzos, utilizando estrategias clásicas dirigidas a ampliar el área de crecimiento de Lmj y garantizar su conservación, han tenido un éxito limitado. El trabajo que se presenta está dirigido a mejorar el conocimiento de la ecología de Lmj, en particular la interacción simbiótica que establece con bacterias del suelo denominadas rizobios y se centra en la caracterización fenotípica, filogenética y genómica de esos rizobios. También se investiga la posible contribución de la simbiosis en mejorar la conservación de Lmj. Para este fin, se han estudiado diferentes aspectos que se describen a continuación. El primero objetivo se centró en aislar y estudiar de la diversidad genética de las bacterias endosimbióticas de Lmj. . Se realizó un análisis filogenético de genes esenciales que mostró que las cepas de Lmj pertenecen al género Bradyrhizobium y que presentan una gran diversidad con características fenotípicas y simbióticas diferentes de cepas de Bradyrhizobium que nodulan otras especies de lupinos nativos de España (cepas ISLU). Las cepas estudiadas se dividieron en dos grupos (Clado I y Clado II). El Clado I, incluye a las cepas Lmj, definiendo un nuevo linaje, filogenéticamente relacionado con otras especies de Bradyrhizobium, como B. jicamae y B. elkanii. El Clado II contiene cepas ISLU relacionadas con cepas de B. canariense y B. japonicum que establecen simbiosis con lupinos de suelos ácidos. Otro análisis filogenético basado en genes simbióticos, distribuyó las cepas de Lmj en sólo dos grupos diferentes. La singularidad y gran diversidad de estas cepas en una pequeña área geográfica, hacen de este, un atractivo sistema para el estudio de la evolución y adaptación de las bacterias simbióticas a su respectiva planta huésped. Adicionalmente, se estudio la presencia de bacterias capaces de nodular Lmj en suelos básicos de Chiapas, México. Sorprendentemente, estos suelos contienen bacterias capaces establecer interacciones simbióticas eficientes con Lmj en ensayos de invernadero. A continuación se investigó la taxonomía de los endosimbiontes de Lmj analizando la secuencia de cuatro genes esenciales (16S rRNA, recA, glnII y atpD) y el promedio de identidad de nucleótidos de genomas completos de algunas cepas representativas de la diversidad (ANIm). Se identificaron nuevas especies de Bradyrhizobium dentro del Clado I y se definió una de ellas: 'Bradyrhizobium valentinum' sp. nov (cepa tipo LmjM3T = CECT 8364T, LMG 2761T). También se abordó cómo conservar Lmj en su hábitat natural mediante inoculación con alguna de las cepas aisladas. Se demostró la ausencia de bacterias capaces de nodular Lmj en suelos rojos alcalinos o ‘‘terra rossa’’ de la Península Ibérica y Baleares. Dos cepas, altamente eficientes en cuanto a la fijación de nitrógeno, LmjC y LmjM3T, fueron seleccionadas para ser empleadas como inoculantes. Dos experimentos de campo llevados a cabo en años consecutivos en áreas con características edafoclimáticas similares a las que presentan las poblaciones de Lmj, lograron la reproducción exitosa de la planta. Se concluyó que un ciclo reproductivo exitoso de Lmj es absolutamente dependiente de la inoculación con sus simbiontes naturales y que la simbiosis debe ser considerada un factor esencial en estrategias de conservación de leguminosas en peligro. La obtención de varias secuencias genómicas de cepas aisladas de Lmj y de otras cepas de Bradyrhizobium reveló una alta similitud entre los genomas de las cepas del Clado I, y permitió la identificación de cinco posibles nuevas especies. Además, se estudiaron tres agrupaciones de genes relacionados con la simbiosis (nod, nif y fix) definiendo un nuevo linaje para las cepas de Lmj, diferente del symbiovar “genistearum” de B. canariense y B. japonicum. La baja diversidad encontrada en el análisis filogenético de los genes simbióticos contrasta con la gran diversidad asociada a genes esenciales. La presencia de plásmidos en cepas del género Bradyrhizobium ha sido descrita en muy pocas ocasiones, sin embargo el análisis de la secuencia genómica de la cepa ISLU101, aislada de Lupinus angustifolius, reveló la presencia de un origen de replicación extracromosómico homólogo al operón repABC, presente en el plásmido de Bradyrhizobium sp BTAi1. Gracias a esta secuencia se identificaron genes homólogos en 19 de 72 cepas ISLU. Filogenéticamente, las secuencias de repABC se agruparon en un grupo monofilético con las de pBTAi1 y separadas de los rizobios de crecimiento rápido. Finalmente, se identificaron sistemas de secreción de proteínas de tipo III (T3SS) en nueve genomas de cepas de Lmj. Los T3SS pueden inyectar proteínas efectoras al interior de células vegetales. Su presencia en rizobios se ha relacionado con la gama de hospedador que pueden nodular y puede tener un efecto beneficioso, neutro o perjudicial en la simbiosis. Los T3SS de las cepas de Lmj codifican para una proteína efectora similar a NopE, un efector dependiente de T3SS descrito en B. diazoefficiens USDA 110T. La proteína NopE de la cepa LmjC se ha caracterizado bioquímicamente. ABSTRACT Lupinus mariae-josephae (Lmj) is a lupine species endemic of a unique small area in Valencia region (Eastern Spain) where the lupine plants thrive in alkaline-limed soils, which preferentially grow in acid or neutral soils. This is the type of soils native lupines of Spain. When this work was initiated, the extension of the endemic area of Lmj was of about 700 squared kilometers confined to the Valencia province. In this area, Lmj thrives in small, isolated patches containing a reduced number of plants, and points to an endemism that can easily became endangered or extinct. Consequently, the Valencia Community authorities gave a ‘‘microreserve” status for conservation of the species. All efforts, using classical strategies directed to extend the area of Lmj growth and ensure its conservation have been so far unsuccessful. The work presented here is directed to improve our knowledge of Lmj ecology and it is centered in the characterization of the rhizobial symbiosis by phenotypic, phylogenetic and genomic analysis as well as in investigate the potential contribution of the symbiosis to improve its conservation. To this end, five different topics have been studied, and results are briefly described here. Extensive details can be followed en the attached, published articles. The first topic deals with the indigenous rhizobial symbionts of the Lmj endemism, and its genetic diversity was investigated. The Lmj root symbionts belong to the Bradyrhizobium genus, and phylogenetic analysis based on core genes identified a large diversity of Bradyrhizobium strains with phenotypic and symbiotic characteristics different from rhizobia nodulating other Lupinus spp. native of Spain. The strains were split in two clades. Clade II contained strains close to classical B. canariense and B. japonicum lineages that establish symbioses with lupines in acid soils of the Mediterranean area. Clade I included Lmj strains that define a new lineage, close to other Bradyrhizobium species as B. jicamae and B. elkanii. The phylogenetic analysis based on symbiotic genes identified only two distinct clusters. The singularity and large diversity of these strains in such a small geographical area makes this an attractive system for studying the evolution and adaptation of the rhizobial symbiont to the plant host. Additionally, the presence of bacteria able to nodulate Lmj in basic soils from Chiapas, Mexico was investigated. Surprisingly, these soils contain bacteria able to effectively nodulate and fix nitrogen with Lmj plants in greenhouse assays. In the second topic, the taxonomic status of the endosymbiotic bacteria of Lmj from Valencia endemism and Chiapas was investigated. Results from phylogenetic analysis of core genes and Average Nucleotide Identity (ANIm) using draft genomic sequences identified new Bradyrhizobium species within strains of Clade I of Lmj endosymbiotic bacteria. Only one of these potentially new species has been defined, meanwhile the others are under process of characterization. The name ‘Bradyrhizobium valentinum’ sp. nov. was proposed for the defined species (type strain LmjM3T= CECT 8364T, LMG 2761T). The third topic was directed to conservation of endangered Lmj in its natural habitat. The relevant conclusion of this experimentation is that the symbiosis should be considered as a relevant factor in the conservation strategies for endangered legumes. First, we showed absence of bacteria able to nodulate Lmj in all the inspected ‘‘terra rossa’’ or alkaline red soils of the Iberian Peninsula and Balearic Islands. Then, two efficient nitrogen fixing strains with Lmj plants, LmjC and LmjM3T, were selected as inoculum for seed coating. Two planting experiments were carried out in consecutive years under natural conditions in areas with edapho-climatic characteristics identical to those sustaining natural Lmj populations, and successful reproduction of the plant was achieved. The relevant conclusion from these assays was that the successful reproductive cycle was absolutely dependent on seedling inoculation with effective bradyrhizobia The forth topic deep into the analysis of the genomic of Lmj representative strains. To this end, draft genomic sequences of selected Lmj strains and type strains of Bradyrhizobium spp. were assembled. The comparison analysis of the draft genomic sequences of Lmj strains and related Bradyrhizobium species grouped in Clade I, revealed a high genomic homology among them, and allowed the definition of five potentially new species of Lmj nodulating bacteria. Also, based on the available draft genomic sequences, only three clusters of nod, fix and nif genes from Lmj strains were identified and showed to define a new symbiotic lineage, distant from that of B. canariense and B. japonicum bv. genistearum. The low diversity exhibited by the phylogenetic analysis of symbiotic genes contrast with the large diversity of strains as regards the housekeeping genes analyzed. Besides, the genomic analysis of a Lupinus angustifolius strain ISLU101, revealed the presence of an extrachromosomal replication origin homologous to repABC cluster from plasmid present in Bradyrhizobium spp BTAi1. This repABC cluster gene sequence allowed the identification of extrachromosomic replication origin in 19 out of 72 Bradyrhizobium strains from Lupinus spp., a highly significant result since the absence of plasmids in the Bradyrhizobium genus was traditionally assumed. The repABC gene sequences of these strains grouped them in a unique monophyletic group, related to B. sp. BTAi1 plasmid, but differentiated from the repABC gene cluster of plasmids in fast growing rhizobium strains. The last topic was focused on characterization of type III secreted effectors present in Lmj endosymbiotic bacteria. Type III secretion systems (T3SS) are specialized protein export machineries which can deliver effector proteins into plant cells. The presence of T3SS in rhizobia has frequently been related to the symbiotic nodulation host-range and may have a beneficial or detrimental effect on the symbiosis with legumes. In this context, the presence of T3SS in genomes of nine Lmj strains was investigated, and it was shown the presence of clusters encoding NopE type III-secreted protein similar to the NopE1 and NopE2 of B. diazoefficiens USDA 110T. The putative NopE protein of LmjC strain is at present being characterized regarding its structure and function.

The ARS309 chromosomal replicator of Saccharomyces cerevisiae depends on an exceptional ARS consensus sequence

Autonomously replicating sequence (ARS) elements, which function as the cis-acting chromosomal replicators in the yeast Saccharomyces cerevisiae, depend upon an essential copy of the 11-bp ARS consensus sequence (ACS) for activity. Analysis of the chromosome III replicator ARS309 unexpectedly revealed that its essential ACS differs from the canonical ACS at two positions. One of the changes observed in ARS309 inactivates other ARS elements. This atypical ACS binds the origin recognition complex efficiently and is required for chromosomal replication origin activity. Comparison of the essential ACS of ARS309 with the essential regions of other ARS elements revealed an expanded 17-bp conserved sequence that efficiently predicts the essential core of ARS elements.

Expression cloning of cDNA encoding a human β-1,3-N-acetylglucosaminyltransferase that is essential for poly-N-acetyllactosamine synthesis

The structure and biosynthesis of poly-N-acetyllactosamine display a dramatic change during development and oncogenesis. Poly-N-acetyllactosamines are also modified by various carbohydrate residues, forming functional oligosaccharides such as sialyl Lex. Herein we describe the isolation and functional expression of a cDNA encoding β-1,3-N-acetylglucosaminyltransferase (iGnT), an enzyme that is essential for the formation of poly-N-acetyllactosamine. For this expression cloning, Burkitt lymphoma Namalwa KJM-1 cells were transfected with cDNA libraries derived from human melanoma and colon carcinoma cells. Transfected Namalwa cells overexpressing the i antigen were continuously selected by fluorescence-activated cell sorting because introduced plasmids containing Epstein–Barr virus replication origin can be continuously amplified as episomes. Sibling selection of plasmids recovered after the third consecutive sorting resulted in a cDNA clone that directs the increased expression of i antigen on the cell surface. The deduced amino acid sequence indicates that this protein has a type II membrane protein topology found in almost all mammalian glycosyltransferases cloned to date. iGnT, however, differs in having the longest transmembrane domain among glycosyltransferases cloned so far. The iGnT transcript is highly expressed in fetal brain and kidney and adult brain but expressed ubiquitously in various adult tissues. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate that the cDNA encodes iGnT, the enzyme responsible for the formation of GlcNAcβ1 → 3Galβ1 → 4GlcNAc → R structure and poly-N-acetyllactosamine extension.

Histone H1 Reduces the Frequency of Initiation in Xenopus Egg Extract by Limiting the Assembly of Prereplication Complexes on Sperm Chromatin

Somatic histone H1 reduces both the rate and extent of DNA replication in Xenopus egg extract. We show here that H1 inhibits replication directly by reducing the number of replication forks, but not the rate of fork progression, in Xenopus sperm nuclei. Density substitution experiments demonstrate that those forks that are active in H1 nuclei elongate to form large tracts of fully replicated DNA, indicating that inhibition is due to a reduction in the frequency of initiation and not the rate or extent of elongation. The observation that H1 dramatically reduces the number of replication foci in sperm nuclei supports this view. The establishment of replication competent DNA in egg extract requires the assembly of prereplication complexes (pre-RCs) on sperm chromatin. H1 reduces binding of the pre-RC proteins, XOrc2, XCdc6, and XMcm3, to chromatin. Replication competence can be restored in these nuclei, however, only under conditions that promote the loss of H1 from chromatin and licensing of the DNA. Thus, H1 inhibits replication in egg extract by preventing the assembly of pre-RCs on sperm chromatin, thereby reducing the frequency of initiation. These data raise the interesting possibility that H1 plays a role in regulating replication origin use during Xenopus development.

A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening

An improved mammalian two-hybrid system designed for interaction trap screening is described in this paper. CV-1/EBNA-1 monkey kidney epithelial cells expressing Epstein–Barr virus nuclear antigen 1 (EBNA-1) were stably transfected with a reporter plasmid for GAL4-dependent expression of the green fluorescent protein (GFP). A resulting clone, GB133, expressed GFP strongly when transfected transiently with transcriptional activators fused to GAL4 DNA-binding domain with minimal background GFP expression. GB133 cells maintained plasmids containing the OriP Epstein–Barr virus replication origin that directs replication of plasmids in mammalian cells in the presence of the EBNA-1 protein. GB133 cells transfected stably with a model bait expressed GFP when further transfected transiently with an expression plasmid for a known positive prey. When the bait-expressing GB133 cells were transfected transiently with an OriP-containing expression plasmid for the positive prey together with excess amounts of empty vector, cells that received the positive prey were readily identified by green fluorescence in cell culture and eventually formed green fluorescent microcolonies, because the prey plasmid was maintained by the EBNA-1/Ori-P system. The green fluorescent microcolonies were harvested directly from the culture dishes under a fluorescence microscope, and total DNA was then prepared. Prey-encoding cDNA was recovered by PCR using primers annealing to the vector sequences flanking the insert-cloning site. This system should be useful in mammalian cells for efficient screening of cDNA libraries by two-hybrid interaction.

Integration of multiple repeats of geminiviral DNA into the nuclear genome of tobacco during evolution.

Integration of viral DNA into the host nuclear genome, although not unusual in bacterial and animal systems, has surprisingly not been reported for plants. We have discovered geminvirus-related DNA (GRD) sequences, in the form of distinct sets of multiple direct repeats comprising three related repeat classes, situated in a unique locus in the Nicotiana tabacum (tobacco) nuclear genome. The organization of these sequences is similar or identical in eight different tobacco cultivars we have examined. DNA sequence analysis reveals that each repeat has sequences most resembling those of the New World geminiviral DNA replication origin plus the adjacent AL1 gene, encoding the viral replication protein. We believe these GRD sequences originated quite recently in Nicotiana evolution through integration of geminiviral DNA by some combination of the processes of illegitimate recombination, amplification, deletions, and rearrangements. These events must have occurred in plant tissue that was subsequently able to contribute to meristematic tissue yielding gametes. GRD may have been retained in tobacco by selection or by random fixation in a small evolving population. Although we cannot detect transcription of these sequences, this does not exclude the possibility that they may originally have been expressed.

Unbalanced R-loops and micronuclei induced by DNA topoisomerase I poisons in cancer cells

Topoisomerase I (Top1) poisons are among the most clinically-effective drugs used for colon, ovary and lung cancers. Unpublished data from our lab have recently revealed that the structurally-unrelated Top1 poisons, Camptothecin (CPT) and Indimitecan (LMP776), induce the formation of micronuclei (MNi) in human cancer cells. In addition, MNi trigger an innate immune gene response by stimulating the cGAS/STING pathway. As the mechanisms of MNi formation are not fully determined, our aim is here to establish how MNi form after Top1 poisoning. Using immunofluorescence assays and EdU labelling of nascent DNAs, our results show that, after 24 hours of recovery, a short treatment with sub-cytotoxic doses of Top1 poisons induces the formation of MNi that do not contain newly synthetized (EdU+) DNA. We also saw that Top1 poisons delay replication machinery reducing EdU incorporation and produce significant levels of the damage markers γH2AX and p53BP1 in S-phase cells but not in G1 and G2/M cells. The results also show that MNi formation is dependent on R-loops, as RNaseH1 overexpression markedly reduces Top1 induced MNi. Genome-wide mapping of R-loops by DRIP-seq technique revealed that R-loop levels are both decreased and increased by CPT. In particular, increased R-loops are mainly found at active genes and always overlapped with Top1cc sites. We also found that increased R-loops overlap with lamina-associated chromatin domains while decreased R-loops correlate with replication origin sites. Overall, our data are consistent with the formation of MNi due to R-loop increase and under-replication at specific regions caused by Top1 poisons. These results will eventually help in developing new strategies for effective personalized interventions by using Top1-targeted compounds as immuno-modulators in cancer patients.

Targeting of DNA polymerase to the adenovirus origin of DNA replication by interaction with nuclear factor I.

Efficient initiation by the DNA polymerase of adenovirus type 2 requires nuclear factor I (NFI), a cellular sequence-specific transcription factor. Three functions of NFI--dimerization, DNA binding, and activation of DNA replication--are colocalized within the N-terminal portion of the protein. To define more precisely the role of NFI in viral DNA replication, a series of site-directed mutations within the N-terminal domain have been generated, thus allowing the separation of all three functions contained within this region. Impairment of the dimerization function prevents sequence-specific DNA binding and in turn abolishes the NFI-mediated activation of DNA replication. NFI DNA-binding activity, although necessary, is not sufficient to activate the initiation of adenovirus replication. A distinct class of NFI mutations that abolish the recruitment of the viral DNA polymerase to the origin also prevent the activation of replication. Thus, a direct interaction of NFI with the viral DNA polymerase complex is required to form a stable and active preinitiation complex on the origin and is responsible for the activation of replication by NFI.

An initial ATP-independent step in the unwinding of a herpes simplex virus type I origin of replication by a complex of the viral origin-binding protein and single-strand DNA-binding protein

Using a spectrophotometric assay that measures the hyperchromicity that accompanies the unwinding of a DNA duplex, we have identified an ATP-independent step in the unwinding of a herpes simplex virus type 1 (HSV-1) origin of replication, Oris, by a complex of the HSV-1 origin binding protein (UL9 protein) and the HSV-1 single-strand DNA binding protein (ICP8). The sequence unwound is the 18-bp A + T-rich segment that links the two high-affinity UL9 protein binding sites, boxes I and II of Oris. P1 nuclease sensitivity of Oris and single-strand DNA-dependent ATPase measurements of the UL9 protein indicate that, at 37°C, the A + T-rich segment is sufficiently single stranded to permit the binding of ICP8. Binding of the UL9 protein to boxes I and II then results in the formation of the UL9 protein–ICP8 complex, that can, in the absence of ATP, promote unwinding of the A + T-rich segment. On addition of ATP, the helicase activity of the UL9 protein–ICP8 complex can unwind boxes I and II, permitting access of the replication machinery to the Oris sequences.

The Ku-like protein from Saccharomyces cerevisiae is required in vitro for the assembly of a stable multiprotein complex at a eukaryotic origin of replication.

We have previously shown that three distinct DNA-binding activities, in crude form, are necessary for the ATP-dependent assembly of a specific and stable multiprotein complex at a yeast origin of replication. Here we show the purification of one of these DNA binding activities, referred to as origin binding factor 2 (OBF2). The purified protein is a heterodimer composed of two polypeptides with molecular mass values of 65 and 80 kDa as determined by SDS/PAGE. Purified OBF2 not only binds DNA but also supports the formation of a protein complex at essential sequences within the ARS121 origin of replication. Interestingly, OBF2 binds tightly and nonspecifically to both duplex DNA and single-stranded DNA. The interaction with duplex DNA occurs at the termini. N-terminal sequencing of the 65-kDa subunit has revealed that this polypeptide is identical to the previously identified HDF1 peptide, a yeast homolog of the small subunit of the mammalian Ku autoantigen. Although the potential involvement of Ku in DNA metabolic events has been proposed, this is the first requirement for a Ku-like protein in the assembly of a protein complex at essential sequences within a eukaryotic origin of replication.

A single-stranded DNA binding protein binds the origin of replication of the duplex kinetoplast DNA.

Replication of the kinetoplast DNA (kDNA) minicircle of trypanosomatids initiates at a conserved 12-nt sequence, 5'-GGGGTTGGTGTA-3', termed the universal minicircle sequence (UMS). A sequence-specific single-stranded DNA-binding protein from Crithidia fasciculata binds the heavy strand of the 12-mer UMS. Whereas this UMS-binding protein (UMSBP) does not bind a duplex UMS dodecamer, it binds the double-stranded kDNA minicircle as well as a duplex minicircle fragment containing the origin-associated UMS. Binding of the minicircle origin region by the single-stranded DNA binding protein suggested the local unwinding of the DNA double helix at this site. Modification of thymine residues at this site by KMnO4 revealed that the UMS resides within an unwound or otherwise sharply distorted DNA at the minicircle origin region. Computer analysis predicts the sequence-directed curving of the minicircle origin region. Electrophoresis of a minicircle fragment containing the origin region in polyacrylamide gels revealed a significantly lower electrophoretic mobility than expected from its length. The fragment anomalous electrophoretic mobility is displayed only in its native conformation and is dependent on temperature and gel porosity, indicating the local curving of the DNA double helix. We suggest that binding of UMSBP at the minicircle origin of replication is possible through local unwinding of the DNA double helix at the UMS site. It is hypothesized here that this local melting is initiated through the untwisting of unstacked dinucleotide sequences at the bent origin site.

In vitro cleavage and joining at the viral origin of replication by the replication initiator protein of tomato yellow leaf curl virus.

Replication of the single-stranded DNA genome of geminiviruses occurs via a double-stranded intermediate that is subsequently used as a template for rolling-circle replication of the viral strand. Only one of the proteins encoded by the virus, here referred to as replication initiator protein (Rep protein), is indispensable for replication. We show that the Rep protein of tomato yellow leaf curl virus initiates viral-strand DNA synthesis by introducing a nick in the plus strand within the nonanucleotide 1TAATATT decreases 8AC, identical among all geminiviruses. After cleavage, the Rep protein remains bound to the 5' end of the cleaved strand. In addition, we show that the Rep protein has a joining activity, suggesting that it acts as a terminase, thus resolving the nascent viral single strand into genome-sized units.