Biological Macromolecules Laboratory • Maeshima Group

Pyrrole-Imidazole (PI) polyamides bind to the minor groove of DNA in a sequence-specific manner. Our previous studies have demonstrated that a synthesized tandem hairpin PI polyamide (TH59) can target human telomere sequences (TTAGGG)n under mild conditions and can serve as a new probe for studying human telomeres. In the recently published two papers, we have improved specificity of the tandem hairpin PI polyamide by optimization of the connecting region (hinge region) of the tandem hairpin parts [HPTH59-b in Fig. A and paper (i)] and by introducing additional hairpin part [TT59 in Fig. B and paper (ii)]. The HPTH59-b and TT59 have higher specificity to recognize human telomeric repeats than the previous ones and stain telomeres in chemically fixed human cells with lower background signal.

(A) Schematic structure of HPTH59-b. The optimized chemical structure of hinge segment is shown.
(B) Left, schematic representation of the tandem trimer PI polyamide TT59, which binds to 18bp of the human telomere sequence. Right, the telomere regions of chromosome ends are labeled with TT59 (green). DNA stain (blue).

The Crafoord Prize(Royal Swedish Academy of Sciences)

Biological Macromolecules Laboratory • Maeshima Group

Genetic information, which is stored in the long strand of genomic DNA as chromatin, must be scanned and read out by various transcription factors. First, gene-specific transcription factors, which are relatively small (~50 kDa), scan the genome and bind regulatory elements. Such factors then recruit general transcription factors, Mediators, RNA polymerases, nucleosome remodellers, and histone modifiers, most of which are large protein complexes of 1–3 MDa in size. Here, we propose a new model for the functional significance of the size of transcription factors (or complexes) for gene regulation of chromatin domains. Recent findings suggest that chromatin consists of irregularly folded nucleosome fibres (10 nm fibres) and forms numerous condensed domains (e.g., topologically associating domains)(blue balls in Fig). Although the flexibility and dynamics of chromatin allow repositioning of genes within the condensed domains, the size exclusion effect of the domain may limit accessibility of DNA sequences by transcription factors. We used Monte Carlo computer simulation to determine the physical size limit of transcription factors that can enter condensed chromatin domains. Small gene-specific transcription factors can penetrate into the chromatin domains and search their target sequences, whereas large transcription complexes cannot enter the domain (Fig a). Due to this property, once a large complex binds its target site via gene-specific factors it can act as a “buoy” to keep the target region on the surface of the condensed domain (Fig b) and maintain transcriptional competency (Fig c). This size-dependent specialisation of target-scanning and surface-tethering functions could provide novel insight into the mechanisms of various DNA transactions, such as DNA replication and repair/recombination.

“Buoy” Model for Transcriptional Regulation
Condensed chromatin domains are shown with blue nucleosomes.
(a) The small transcription factors in yellow can move in the condensed chromatin domain but not large transcription complexes (in green). (b) The small transcription factor bound to the target region (red nucleosomes) and can recruit large transcription complexes when it is relocated on the domain surface. Binding of large transcription complexes (green) keeps the transcriptional region (red nucleosomes) on the domain surface like a ‘buoy’. (c) With other large complexes such as nucleosome remodeler or histone modifier, stable transcription is maintained.

超分子構造研究室・白木原研究室

Duplex DNA is generally unwound by protein oligomers prior to replication. The Rep protein of plasmid ColE2-P9 is an essential initiator for plasmid DNA replication. This protein binds the replication origin (Ori) in a sequence specific manner as a monomer and unwinds DNA. We present the crystal structure of the DNA-binding domain of Rep (E2Rep-DBD) in complex with Ori DNA. The structure unveils the basis for Ori specific recognition by the E2Rep-DBD and also reveals that it unwinds DNA by the concerted actions of its three contiguous structural modules. The structure also shows that the functionally unknown PriCT domain, which forms a compact module, plays a central role in DNA unwinding. The conservation of the PriCT domain in the C-termini of some archaeo-eukaryotic primases, indicates that it likely plays a similar role in these proteins. Thus, this is the first report providing the structural basis for the functional importance of the conserved PriCT domain and also reveals a novel mechanism for DNA unwinding by a single protein.

(A) The ribbon model of the complex consists of E2Rep-DBD and Ori DNA. Elongated fold of E2Rep-DBD enables binding specificity and affinity enough to unwind duplex DNA.
(B) Model for unwinding of the complete Ori by Rep. The PriCT-module plays a central role in unwinding of duplex DNA and stabilizing the single-stranded DNA.