Hugo Maruyama

Molecular mechanisms of DNA end‐loop formation by TRF2

In the telomere region of human chromosomes, the (TTAGGG)n sequence stretches over several kilobases and forms a distinct higher‐order structure with various proteins. Telomere repeat binding factors (TRFs) bind specifically to this sequence and play critical roles in the maintenance of telomere structure and function. Here, we prepared a series of linear DNA carrying a stretch of telomeric sequence ((TTAGGG)n, ∼1.8 (kb) with different end‐structures and observed their higher‐order complexes with TRFs by atomic force microscopy. TRF2 molecules exclusively bound to the telomeric DNA region at several different places simultaneously mainly as a dimer, and often mediated DNA loop formation by forming a tetramer at the root. These multiple‐binding, multimerization and DNA loop formation by TRF2 were observed regardless of the DNA‐end structure (blunt, 3′‐overhanging, telomeric, non‐telomeric). However, when the DNA end carried the telomeric‐3′‐overhanging region, the loop was frequently formed at the end of the DNA. Namely, the TRF2‐mediated DNA loop formation is independent of the end‐structure and the 3′‐overhanging TTAGGG sequence is responsible for the stabilization of the loop. TRF1 also bound to the telomeric DNA as a dimer, but did not mediate DNA loop formation by itself. These results provide a new insight into the molecular mechanism of DNA end‐loop formation by TRFs.

An alternative beads‐on‐a‐string chromatin architecture in Thermococcus kodakarensis

We have applied chromatin sequencing technology to the euryarchaeon Thermococcus kodakarensis, which is known to possess histone‐like proteins. We detect positioned chromatin particles of variable sizes associated with lengths of DNA differing as multiples of 30 bp (ranging from 30 bp to >450 bp) consistent with formation from dynamic polymers of the archaeal histone dimer. T. kodakarensis chromatin particles have distinctive underlying DNA sequence suggesting a genomic particle‐positioning code and are excluded from gene‐regulatory DNA suggesting a functional organization. Beads‐on‐a‐string chromatin is therefore conserved between eukaryotes and archaea but can derive from deployment of histone‐fold proteins in a variety of multimeric forms.

Organismal propagation in the absence of a functional telomerase pathway in Caenorhabditis elegans

To counteract replication‐dependent telomere shortening most eukaryotic cells rely on the telomerase pathway, which is crucial for the maintenance of proliferative potential of germ and stem cell populations of multicellular organisms. Likewise, cancer cells usually engage the telomerase pathway for telomere maintenance to gain immortality. However, in ∼10% of human cancers telomeres are maintained through telomerase‐independent alternative lengthening of telomeres (ALT) pathways. Here, we describe the generation and characterization of C. elegans survivors in a strain lacking the catalytic subunit of telomerase and the nematode telomere‐binding protein CeOB2. These clonal strains, some of which have been propagated for >180 generations, represent the first example of a multicellular organism with canonical telomeres that can survive without a functional telomerase pathway. The animals display the heterogeneous telomere length characteristic for ALT cells, contain single‐stranded C‐circles, a transcription profile pointing towards an adaptation to chronic stress and are therefore a unique and valuable tool to decipher the ALT mechanism.

Transcriptional Repressor TrmBL2 from Thermococcus kodakarensis Forms Filamentous Nucleoprotein Structures and Competes with Histones for DNA Binding in a Salt- and DNA Supercoiling-dependent Manner

Background: TrmBL2 and histones maintain the genome functioning in hyperthermophilic euryarchaeal cells. Results: TrmBL2 forms filaments on DNA through cooperative binding and competes with histones in a salt- and DNA supercoiling-dependent manner. Conclusion: TrmBL2-histone collective behavior dynamically controls DNA organization. Significance: The discovered mechanisms provide insights into the regulation of the genome structure and global transcription profile by TrmBL2 and histones. Architectural DNA proteins play important roles in the chromosomal DNA organization and global gene regulation in living cells. However, physiological functions of some DNA-binding proteins from archaea remain unclear. Recently, several abundant DNA-architectural proteins including histones, Alba, and TrmBL2 have been identified in model euryarchaeon Thermococcus kodakarensis. Although histones and Alba proteins have been previously characterized, the DNA binding properties of TrmBL2 and its interplay with the other major architectural proteins in the chromosomal DNA organization and gene transcription regulation remain largely unexplored. Here, we report single-DNA studies showing that at low ionic strength (<300 mm KCl), TrmBL2 binds to DNA largely in non-sequence-specific manner with positive cooperativity, resulting in formation of stiff nucleoprotein filamentous patches, whereas at high ionic strength (>300 mm KCl) TrmBL2 switches to more sequence-specific interaction, suggesting the presence of high affinity TrmBL2-filament nucleation sites. Furthermore, in vitro assays indicate the existence of DNA binding competition between TrmBL2 and archaeal histones B from T. kodakarensis, which can be strongly modulated by DNA supercoiling and ionic strength of surrounding solution. Overall, these results advance our understanding of TrmBL2 DNA binding properties and provide important insights into potential functions of architectural proteins in nucleoid organization and gene regulation in T. kodakarensis.

A Highlights from MBoC Selection: Histone and TK0471/TrmBL2 form a novel heterogeneous genome architecture in the hyperthermophilic archaeon Thermococcus kodakarensis

This study demonstrates that the chromosome of the hyperthermophilic archaeon Thermococcus kodakarensis is organized into a heterogeneous structure created with histone and a novel protein TK0471/TrmBL2. TK0471/TrmBL2 plays dual roles as a chromosomal protein and as a global transcriptional repressor, and it is conserved in some archaeal and bacterial species.

Identification of disulphide stress–responsive extracytoplasmic function sigma factors in Rothia mucilaginosa

Rothia mucilaginosa is known as a member of commensal bacterial flora in the oral cavity and has received attention as a potential opportunistic pathogen. We previously determined the genomic sequence of R. mucilaginosa DY-18, a clinical strain with biofilm-like structures isolated from an infected root canal of a tooth with persistent apical periodontitis. We found that the DY-18 genome had only two sigma factor genes that encoded the primary and extracytoplasmic function (ECF) sigma factors. Genomic analysis on the available database of R. mucilaginosa ATCC 25296 (a type strain for R. mucilaginosa) revealed that ATCC 25296 has three sigma factors: one primary sigma factor and two ECF sigma factors, one of which was highly homologous to that of DY-18. ECF sigma factors play an important role in the response to environmental stress and to the production of virulence factors. Therefore, we first examined gene-encoding sigma factors on R. mucilaginosa genome in silico. The homologous ECF sigma factors found in strains DY-18 and ATCC 25296 formed a distinct SigH (SigR) clade in a phylogenetic tree and their cognate anti-sigma factor has a HXXXCXXC motif known to respond against disulphide stress. Quantitative reverse transcription polymerase chain reaction (PCR) and microarray analysis showed that the transcriptional levels of sigH were markedly up-regulated under disulphide stress in both strains. Microarray data also demonstrated that several oxidative-stress-related genes (thioredoxin, mycothione reductase, reductase and oxidoreductase) were significantly up-regulated under the diamide stress. On the basis of these results, we conclude that the alternative sigma factor SigH of R. mucilaginosa is a candidate regulator in the redox state.

Complete Genome Sequence of Prevotella intermedia Strain 17-2

ABSTRACT Prevotella intermedia, a Gram-negative black-pigmented anaerobic rod, is frequently isolated from not only periodontal pockets but also purulent infections. We report here the complete genome sequence of P. intermedia strain 17-2, which is a non-exopolysaccharide-producing variant obtained from exopolysaccharide (EPS)-producing P. intermedia strain 17 stock culture.

Exopolysaccharide Productivity and Biofilm Phenotype on Oral Commensal Bacteria as Pathogenesis of Chronic Periodontitis

Abstract : Exopolysaccharide (EPS) productivities in many bacteria have been associated with pathogenicity in mammalian hosts as providing extracellular matrices to form biofilm (Costerton et al., 1995). Bacteria assuming biofilm-forming capacity have enormous advantages in establishing persistent infections (Costerton et al., 1999). Chronic periodontitis is caused by dental plaque known as a complex biofilm which consists of several hundred different species of bacteria (Chen, 2001; Socransky and Haffajee, 2002; Lovegrove, 2004). While sucrose-derived homopolysaccharides are well known substrates which mediate adhesion of bacteria to the tooth surface and co-aggregation interactions between species of oral bacteria in the dental plaque (Russell, 2009), recent studies suggest that each bacterium produces distinctive EPS components in a sucrose-independent manner and can form so called single species biofilm (Branda et al., 2005). In the oral cavity, several species of oral bacteria are known to produce their own EPS with this manner (Okuda et al., 1987; Dyer and Bolton, 1985; Kaplan et al., 2004; Yamane et al., 2005; Yamanaka et al., 2009; Yamanaka et al., 2010). In this chapter, we will describe a possibility that a single species biofilm in the oral cavity can cause persistent chronic periodontitis along with the importance of dental plaque formation and maturation with sucrose-derived polysaccharides.

Gas gangrene in the deep spaces of the head and neck visualized on computed tomography images

Cellulitis accompanied by gas gangrene is a rapidly-spreading and potentially fatal infection. Here, we present a case of gas gangrene in the deep spaces of the head and neck in an elderly woman, diagnosed by computed tomography (CT). An 86-year-old woman with Alzheimer’s disease, hypertension, hyperlipidemia, and osteoporosis was referred to our institute by her local dentist. The patient exhibited trismus caused by severe swelling in the left submandibular area. CT images of the head and neck area showed swelling of the cervical tissue with air in the parapharyngeal and masticator spaces. She was treated with antibiotics, followed by drainage. Although the therapy was continued, the patient died from a cardiac complication on hospital day 42. Our case highlights the usefulness of CT for diagnosing gas gangrene in the deep spaces of the head and neck in a woman with Alzheimer’s disease.

Atomic Force Microscopy Imaging and Analysis of Prokaryotic Genome Organization

This protocol describes the application of atomic force microscopy for structural analysis of the prokaryotic and organellar nucleoids. It is based on a simple cell manipulation procedure that enables step-wise dissection of the nucleoid. The procedure includes (1) on-substrate-lysis of cells, and (2) enzyme treatment, followed by atomic force microscopy. This type of dissection analysis permits analysis of nucleoid structure ranging from the fundamental units assembled on DNA to higher order levels of organization. The combination with molecular-genetic and biochemical techniques further permits analysis of the functions of key nucleoid factors relevant to signal-induced structural re-organization or building up of basic structures, as seen for Dps in Escherichia coli, and TrmBL2 in Thermococcus kodakarensis. These systems are described here as examples of the successful application of AFM for this purpose. Moreover, we describe the procedures needed for quantitative analysis of the data.