Kodai Hara

REV1 is a eukaryotic error-prone DNA polymerase belonging to the Y-family, with a central role in translesion DNA synthesis (TLS) to continue DNA replication even in the presence of DNA damage in the template strand. TLS is stimulated by monoubiquitination of proliferating cell nuclear antigen (PCNA), a toroidal-shaped protein functioning as a scaffold for DNA polymerases and repair enzymes. Mammals possess four types of Y-family DNA polymerases: Pol η, Pol κ, Pol ι and REV1. Among those, Pol η, Pol κ and Pol ι interact with PCNA through PCNA-binding motifs, low-affinity variants of PCNA-interacting protein box (PIP-box). To date, several studies have reported that REV1 interacts with PCNA, but identified PCNA-binding regions are inconsistent; therefore, a structural basis for interaction between REV1 and PCNA also remains unclear. Here, we identified a signature sequence conserved within vertebrates REV1 responsible for PCNA-binding. Furthermore, we unveiled a mechanism underlying the physical interaction between the PCNA-binding motif of human REV1 and PCNA by X-ray crystallography, thus revealing that REV1 binds to PCNA through a PIP-box variant located in the C-terminal side of the little finger domain. Our study provides a convincing answer for a long-standing controversy regarding the physical interaction between REV1 and PCNA.

Tyrosine phenol-lyase (TPL), which is expressed in intestinal bacteria, catalyzes the formation of phenol from the substrate L-Tyr. Bacterial metabolite phenol and the sulfate conjugate (phenyl sulfate) are known as a type of uremic toxins, some of which exert cytotoxicity. Therefore, pathologically elevated phenol and phenyl sulfate levels are strongly implicated in the etiology and outcome of uremia. In this study, we explored the inhibitory effects of dietary polyphenols on TPL-catalyzed phenol production using a TPL activity assay. Quercetin, one of the most popular polyphenols, exhibited the strongest inhibitory activity (K_i = 19.9 µM). Quercetin competitively inhibited TPL, and its activity was stronger than that of a known TPL inhibitor (Tyr analog; 2-aza-Tyr, K_i = 42.0 µM). Additionally, quercetin significantly inhibited phenol production in TPL-expressing bacterial cultures (Morganella morganii and Citrobacter koseri) and Tyr-rich (5%) diet-fed C57BL/6J mouse feces. Our findings suggest that quercetin is the most promising polyphenol for reducing phenol levels. Because quercetin has a low gastrointestinal absorption rate, TPL inhibition in the intestinal tract by quercetin may be an effective strategy for treating uremia.

The sliding DNA clamp is a ring-shaped protein that encircles DNA within its central channel. It binds to multiple proteins, such as DNA polymerases and DNA repair enzymes, and stimulates their enzymatic activities, thereby playing a crucial role in cell survival and proliferation. Accordingly, the bacterial clamp DnaN is considered to be a promising target for bacterial infection therapy. In this regard, 3D structures of DnaN from pathogenic bacteria are essential for the development of chemical compounds with antimicrobial activity. Here, the crystal structure of DnaN from a Gram-positive bacterium Clostridioides difficile, a human pathogen causing infectious diarrhoea, has been determined at 2.13 Å resolution. A comparison of the structures of DnaN from other bacteria indicates that the structural features of DnaN in terms of overall organization are essentially conserved within Gram-positive and Gram-negative bacteria. However, DnaN from C. difficile has structural differences in the potential binding pocket for partner proteins, implying a non-conventional interaction with its binding partners. Our findings will provide insight into the development of new therapies for C. difficile infection.

DNA sliding clamps are widely conserved in all living organisms and play crucial roles in DNA replication and repair. Each DNA sliding clamp is a doughnut-shaped protein with a quaternary structure that encircles the DNA strand and recruits various factors involved in DNA replication and repair, thereby stimulating their biological functions. Eukaryotes have two types of DNA sliding clamp, proliferating cell nuclear antigen (PCNA) and RAD9-RAD1-HUS1 (9-1-1). The homo-trimer PCNA physically interacts with multiple proteins containing a PCNA-interacting protein box and/or AlkB homologue 2 PCNA-interacting motif. The two motifs bind to PCNA by a similar mechanism; in addition, the bound PCNA structure is similar, implying a universality of PCNA interactions. In contrast to PCNA, 9-1-1 is a hetero-trimer composed of RAD9, RAD1 and HUS1 subunits. Although 9-1-1 forms a trimeric ring structure similar to PCNA, the C-terminal extension of the RAD9 is intrinsically unstructured. Based on the structural similarity between PCNA and 9-1-1, the mechanism underlying the interaction of 9-1-1 with its partners was thought to be analogous to that of PCNA. Unexpectedly, however, the recent structure of the 9-1-1 ring bound to a partner has revealed a novel interaction distinct from that of PCNA, potentially providing a new principle for molecular interactions on DNA sliding clamps.

The N-terminal region of the stomatin operon partner protein (STOPP) PH1510 (1510-N) from the hyperthermophilic archaeonPyrococcus horikoshiiis a serine protease with a catalytic Ser–Lys dyad (Ser97 and Lys138) and specifically cleaves the C-terminal hydrophobic region of the p-stomatin PH1511. In a form of human hemolytic anemia known as hereditary stomatocytosis, stomatin is deficient in the erythrocyte membrane owing to mis-trafficking. Stomatin is thought to act as an oligomeric scaffolding protein to support cell membranes. The cleavage of stomatin by STOPP might be involved in a regulatory system. Several crystal structures of 1510-N have previously been determined: the wild type, the K138A mutant and its complex with a substrate peptide. Here, the crystal structure of the S97A mutant of 1510-N (1510-N S97A) was determined at 2.25 Å resolution. The structure contained two 1510-N S97A molecules in the asymmetric unit. On the superposition of one monomer of the 1510-N S97A and wild-type dimers, the S97A C^αatom of the other monomer of 1510-N S97A deviated by 23 Å from that of the wild type. This result indicates that 1510-N can greatly change the form of its dimer. Because of crystallographic symmetry in space groupP6₅, a sixfold helical structure is constructed using the 1510-N dimer as a basic unit. This helical structure may be common to STOPP structures.

Condensin I is a multi‐protein complex that plays an essential role in mitotic chromosome assembly and segregation in eukaryotes. It is composed of five subunits: two SMC (SMC2 and SMC4), a kleisin (CAP‐H), and two HEAT‐repeat (CAP‐D2 and CAP‐G) subunits. Although balancing acts of the two HEAT‐repeat subunits have been demonstrated to enable this complex to support the dynamic assembly of chromosomal axes in vertebrate cells, its underlying mechanisms remain poorly understood. Here, we report the crystal structure of a human condensin I subcomplex comprising hCAP‐G and hCAP‐H. hCAP‐H binds to the concave surfaces of a harp‐shaped HEAT‐repeat domain of hCAP‐G. Physical interaction between hCAP‐G and hCAP‐H is indeed essential for mitotic chromosome assembly recapitulated in Xenopus egg cell‐free extracts. Furthermore, this study reveals that the human CAP‐G‐H subcomplex has the ability to interact with not only double‐stranded DNA, but also single‐stranded DNA, suggesting functional divergence of the vertebrate condensin I complex in proper mitotic chromosome assembly.

ABSTRUCT: Condensin I is a multi-protein complex that plays an essential role in mitotic chromosome assembly and segregation in eukaryotes. It is composed of five subunits: two SMC (SMC2 and SMC4), a kleisin (CAP-H) and two HEAT-repeat (CAP-D2 and -G) subunits. Although it has been shown that balancing acts of the two HEAT-repeat subunits enable this complex to support dynamic assembly of chromosomal axes in vertebrate cells, its underlying mechanisms remain poorly understood. Here, we report the crystal structure of a human condensin I subcomplex comprising hCAP-G and hCAP-H. hCAP-H binds to the concave surfaces of a harp-shaped HEAT repeat domain of hCAP-G. A physical interaction between hCAP-G and hCAP-H is indeed essential for mitotic chromosome assembly recapitulated in Xenopus egg cell-free extracts. Furthermore, this study reveals that the human CAP-G-H subcomplex has the ability to interact with not only a double-stranded DNA, but also a single-stranded DNA, implicating potential, functional divergence of the vertebrate condensin I complex in mitotic chromosome assembly.

For a better understanding of protein–inhibitor interactions, we report structural, thermodynamic, and biological analyses of the interactions between S-trityl-l-cysteine (STLC) derivatives and the motor domain of kinesin spindle protein Eg5. Binding of STLC-type inhibitors to Eg5 was enthalpically driven and entropically unfavorable. The introduction of a para-methoxy substituent in one phenyl ring of STLC enhances its inhibitory activity resulting from a larger enthalpy gain possibly due to the increased shape complementarity. The substituent fits to a recess in the binding pocket. To avoid steric hindrance, the substituted STLC is nudged toward the side opposite to the recess, which enhances the interaction of Eg5 with the remaining part of the inhibitor. Further introduction of an ethylene linkage between two phenyl rings enhances Eg5 inhibitory activity by reducing the loss of entropy in forming the complex. This study provides valuable examples of enhancing protein–inhibitor interactions without forming additional hydrogen bonds.