Crystal structure and centromere binding of the plasmid segregation protein ParB from pCXC100

Plasmid pCXC100 from the Gram-positive bacterium Leifsonia xyli subsp. cynodontis uses a type Ib partition system that includes a centromere region, a Walker-type ATPase ParA and a centromere-binding protein ParB for stable segregation. However, ParB shows no detectable sequence homology to any DNA-binding motif. Here, we study the ParB centromere interaction by structural and biochemical approaches. The crystal structure of the C-terminal DNA-binding domain of ParB at 1.4 Å resolution reveals a dimeric ribbon–helix–helix (RHH) motif, supporting the prevalence of RHH motif in centromere binding. Using hydroxyl radical footprinting and quantitative binding assays, we show that the centromere core comprises nine uninterrupted 9-nt direct repeats that can be successively bound by ParB dimers in a cooperative manner. However, the interaction of ParB with a single subsite requires 18 base pairs covering one immediate repeat as well as two halves of flanking repeats. Through mutagenesis, sequence specificity was determined for each position of an 18-bp subsite. These data suggest an unique centromere recognition mechanism by which the repeat sequence is jointly specified by adjacent ParB dimers bound to an overlapped region.     

Centromere binding specificity in assembly of the F plasmid partition complex

The segregation of plasmid F of Escherichia coli is highly reliable. The Sop partition locus, responsible for this stable maintenance, is composed of two genes, sopA and sopB and a centromere, sopC, consisting of 12 direct repeats of 43 bp. Each repeat carries a 16-bp inverted repeat motif to which SopB binds to form a nucleoprotein assembly called the partition complex. A database search for sequences closely related to sopC revealed unexpected features that appeared highly conserved. We have investigated the requirements for specific SopB–sopC interactions using a surface plasmon resonance imaging technique. We show that (i) only 10 repeats interact specifically with SopB, (ii) no base outside the 16-bp sopC sites is involved in binding specificity, whereas five bases present in each arm are required for interactions, and (iii) the A-C central bases contribute to binding efficiency by conforming to a need for a purine–pyrimidine dinucleotide. We have refined the SopB–sopC binding pattern by electro-mobility shift assay and found that all 16 bp are necessary for optimal SopB binding. These data and the model we propose, define the basis of the high binding specificity of F partition complex assembly, without which, dispersal of SopB over DNA would result in defective segregation.     

Genomic Sequence Analysis of Bombyx mori Nucleopolyhedrovirus Isolated from Yunnan Sericulture Region,China

The blood pathogens of grasserie caused by Bombyx mori nucleopolyhedrovirus BmNPV have a serious impact on the sericulture industry. To understand the genetic status of BmNPV endemic strains in the Yunnan sericulture region, the structure and complete genome sequence of BmNPV isolated from Baoshan city of Yunnan Province were described and compared to known strains. The BmNPV-Baoshan isolate was a nucleopolyhedrovirus parasitized in silkworm larvae. Its genome has 128, 452 bp with a G + C content of 40.4%. Phylogenetic analysis clustered the virus with China BmNPV isolates; BmNPV-Baoshan was closely related to BomaNPV-S1 (both strains originated from the same ancestor). BmNPV-Baoshan strain has bro-b gene deletion, hr1 missing 4 repeat units of 30-bp palindrome structure compared to BmNPV-T3 strain. The aim of this study was to elucidate the evolution of the virus further and provide insights for the protection of virus-induced hematologic sepsis.     

Draft Genome Assembly and Annotation for Cutaneotrichosporon dermatis NICC30027, an Oleaginous Yeast Capable of Simultaneous Glucose and Xylose Assimilation

The identification of oleaginous yeast species capable of simultaneously utilizing xylose and glucose as substrates to generate value-added biological products is an area of key economic interest. We have previously demonstrated that the Cutaneotrichosporon dermatis NICC30027 yeast strain is capable of simultaneously assimilating both xylose and glucose, resulting in considerable lipid accumulation. However, as no high-quality genome sequencing data or associated annotations for this strain are available at present, it remains challenging to study the metabolic mechanisms underlying this phenotype. Herein, we report a 39,305,439 bp draft genome assembly for C. dermatis NICC30027 comprised of 37 scaffolds, with 60.15% GC content. Within this genome, we identified 524 tRNAs, 142 sRNAs, 53 miRNAs, 28 snRNAs, and eight rRNA clusters. Moreover, repeat sequences totaling 1,032,129 bp in length were identified (2.63% of the genome), as were 14,238 unigenes that were 1,789.35 bp in length on average (64.82% of the genome). The NCBI non-redundant protein sequences (NR) database was employed to successfully annotate 11,795 of these unigenes, while 3,621 and 11,902 were annotated with the Swiss-Prot and TrEMBL databases, respectively. Unigenes were additionally subjected to pathway enrichment analyses using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Cluster of Orthologous Groups of proteins (COG), Clusters of orthologous groups for eukaryotic complete genomes (KOG), and Non-supervised Orthologous Groups (eggNOG) databases. Together, these results provide a foundation for future studies aimed at clarifying the mechanistic basis for the ability of C. dermatis NICC30027 to simultaneously utilize glucose and xylose to synthesize lipids.     

Identification of high affinity Tbf1p-binding sites within the budding yeast genome

The yeast TBF1 gene is essential for mitotic growth and encodes a protein that binds the human telomere repeats in vitro, although its cellular function is unknown. The sequence of the DNA-binding domain of Tbf1p is more closely related to that of the human telomeric proteins TRF1 and TRF2 than to any yeast protein sequence, yet the functional homologue of TRF1 and TRF2 is thought to be Rap1p. In this study we show that the Tbf1p DNA-binding domain can target the Gal4 transactivation domain to a (TTAGGG)_n sequence inserted in the yeast genome, supporting the model that Tbf1p binds this sub-telomeric repeat motif in vivo. Immunofluorescence of Tbf1p shows a spotty pattern throughout the interphase nucleus and along synapsed chromosomes in meiosis, suggesting that Tbf1p binds internal chromosomal sites in addition to sub-telomeric regions. PCR-assisted binding site selection was used to define a consensus for high affinity Tbf1p-binding sites. Compilation of 50 selected oligonucleotides identified the consensus TAGGGTTGG. Five potential Tbf1p-binding sites resulting from a search of the total yeast genome were tested directly in gel shift assays and shown to bind Tbf1p efficiently in vitro, thus confirming this as a valid consensus for Tbf1p recognition.     

Prod is a novel DNA-binding protein that binds to the 1.686 g/cm(3) 10 bp satellite repeat of Drosophila melanogaster

The proliferation disrupter (prod) gene of Drosophila melanogaster encodes a novel protein associated with centromeric chromosomal regions that is required for chromatin condensation and cell viability. We have examined the binding of the Prod protein to DNA in vitro. Co-immunoprecipitation experiments demonstrate that Prod is a DNA-binding protein that specifically recognizes the 10 bp AGAATAACAT satellite repeat of D.melanogaster. Footprinting experiments show that the protein interacts with a 5–8 bp target sequence in each 10 bp repeat and suggest that it can mediate condensation of this satellite into a superhelix. Gel retardation experiments indicate that Prod does not have a well defined DNA-binding domain and it binds the satellite in a co-operative manner, probably forming Prod multimers. Since Prod localizes to both heterochromatin and euchromatin in vivo, we discuss the possibility that the ability of pre-existing euchromatic proteins to bind DNA in a co-operative manner, might be a prerequisite of satellite compaction and satellite amplification, thereby providing a basic factor in heterochromatin evolution.     

Population-specific recombination sites within the human MHC region

Genetic rearrangement by recombination is one of the major driving forces for genome evolution, and recombination is known to occur in non-random, discreet recombination sites within the genome. Mapping of recombination sites has proved to be difficult, particularly, in the human MHC region that is complicated by both population variation and highly polymorphic HLA genes. To overcome these problems, HLA-typed individuals from three representative populations: Asian, European and African were used to generate phased HLA haplotypes. Extended haplotype homozygosity (EHH) plots constructed from the phased haplotype data revealed discreet EHH drops corresponding to recombination events and these signatures were observed to be different for each population. Surprisingly, the majority of recombination sites detected are unique to each population, rather than being common. Unique recombination sites account for 56.8% (21/37 of total sites) in the Asian cohort, 50.0% (15/30 sites) in Europeans and 63.2% (24/38 sites) in Africans. Validation carried out at a known sperm typing recombination site of 45 kb (HLA-F-telomeric) showed that EHH was an efficient method to narrow the recombination region to 826 bp, and this was further refined to 660 bp by resequencing. This approach significantly enhanced mapping of the genomic architecture within the human MHC, and will be useful in studies to identify disease risk genes.     

Sequence analysis of the insertion element ISH1.8 and of associated structural changes in the genome of phage PhiH of the archaebacterium Halobacterium halobium

We have sequenced the insertion element ISH1.8 which can be present in one or two copies in the genome of phage ΦH of Halobacterium halobium. ISH1.8 is 1895 bp long, has no inverted repeat at its ends, and one only of the two copies is flanked by two 5-bp duplications. An 8-bp sequence composed of 4 bp from each end of ISH1.8 is present in both sites lacking the element. This 8-bp sequence could either be a specific insertion sequence or a part of the element that is left behind upon deletion. The plasmid pΦHL, consisting of the invertible L segment of the phage genome which is, in ΦH2 and ΦH5, flanked by two copies of ISH1.8, contains 112 bp of ISH1.8 and is released from the phage genome by recombination within a direct repeat of 9 bp. This 9-bp sequence (TCCCGCCCT) exists as an inverted repeat in ISH1.8 and therefore as two distinct repeats in phage genomes containing two copies of ISH1.8 in inverted orientation.     

Structural basis of replication origin recognition by the DnaA protein

Escherichia coli DnaA binds to 9 bp sequences (DnaA boxes) in the replication origin, oriC, to form a complex initiating chromosomal DNA replication. In the present study, we determined the crystal structure of its DNA-binding domain (domain IV) complexed with a DnaA box at 2.1 Å resolution. DnaA domain IV contains a helix–turn–helix motif for DNA binding. One helix and a loop of the helix– turn–helix motif are inserted into the major groove and 5 bp (3′ two-thirds of the DnaA box sequence) are recognized through base-specific hydrogen bonds and van der Waals contacts with the C5-methyl groups of thymines. In the minor groove, Arg399, located in the loop adjacent to the motif, recognizes three more base pairs (5′ one-third of the DnaA box sequence) by base-specific hydrogen bonds. DNA bending by ~28° was also observed in the complex. These base-specific interactions explain how DnaA exhibits higher affinity for the strong DnaA boxes (R1, R2 and R4) than the weak DnaA boxes (R3 and M) in the replication origin.     

RNA G-Quadruplexes in the model plant species Arabidopsis thaliana: prevalence and possible functional roles

Tandem stretches of guanines can associate in hydrogen-bonded arrays to form G-quadruplexes, which are stabilized by K⁺ ions. Using computational methods, we searched for G-Quadruplex Sequence (GQS) patterns in the model plant species Arabidopsis thaliana. We found ∼1200 GQS with a G₃ repeat sequence motif, most of which are located in the intergenic region. Using a Markov modeled genome, we determined that GQS are significantly underrepresented in the genome. Additionally, we found ∼43 000 GQS with a G₂ repeat sequence motif; notably, 80% of these were located in genic regions, suggesting that these sequences may fold at the RNA level. Gene Ontology functional analysis revealed that GQS are overrepresented in genes encoding proteins of certain functional categories, including enzyme activity. Conversely, GQS are underrepresented in other categories of genes, notably those for non-coding RNAs such as tRNAs and rRNAs. We also find that genes that are differentially regulated by drought are significantly more likely to contain a GQS. CD-detected K⁺ titrations performed on representative RNAs verified formation of quadruplexes at physiological K⁺ concentrations. Overall, this study indicates that GQS are present at unique locations in Arabidopsis and that folding of RNA GQS may play important roles in regulating gene expression.     

DNA Crossover Motifs Associated with Epigenetic Modifications Delineate Open Chromatin Regions in Arabidopsis

The rate of crossover, the reciprocal exchanges of homologous chromosomal segments, is not uniform along chromosomes differing between male and female meiocytes. To better understand the factors regulating this variable landscape, we performed a detailed genetic and epigenetic analysis of 737 crossover events in Arabidopsis thaliana. Crossovers were more frequent than expected in promoters. Three DNA motifs enriched in crossover regions and less abundant in crossover-poor pericentric regions were identified. One of these motifs, the CCN repeat, was previously unknown in plants. The A-rich motif was preferentially associated with promoters, while the CCN repeat and the CTT repeat motifs were preferentially associated with genes. Analysis of epigenetic modifications around the motifs showed, in most cases, a specific epigenetic architecture. For example, we show that there is a peak of nucleosome occupancy and of H3K4me3 around the CCN and CTT repeat motifs while nucleosome occupancy was lowest around the A-rich motif. Cytosine methylation levels showed a gradual decrease within ∼2 kb of the three motifs, being lowest at sites where crossover occurred. This landscape was conserved in the decreased DNA methylation1 mutant. In summary, the crossover motifs are associated with epigenetic landscapes corresponding to open chromatin and contributing to the nonuniformity of crossovers in Arabidopsis.