Effects of chain length and geometry on the activation of DNA damage bypass by polyubiquitylated PCNA.

Ubiquitylation of the eukaryotic sliding clamp, PCNA, activates a pathway of DNA damage bypass that facilitates the replication of damaged DNA. In its monoubiquitylated form, PCNA recruits a set of damage-tolerant DNA polymerases for translesion synthesis. Alternatively, modification by K63-linked polyubiquitylation triggers a recombinogenic process involving template switching. Despite the identification of proteins interacting preferentially with polyubiquitylated PCNA, the molecular function of the chain and the relevance of its K63-linkage are poorly understood. Using genetically engineered mimics of polyubiquitylated PCNA, we have now examined the properties of the ubiquitin chain required for damage bypass in budding yeast. By varying key parameters such as the geometry of the junction, cleavability and capacity for branching, we demonstrate that either the structure of the ubiquitin-ubiquitin junction or its dynamic assembly or disassembly at the site of action exert a critical impact on damage bypass, even though known effectors of polyubiquitylated PCNA are not strictly linkage-selective. Moreover, we found that a single K63-junction supports substantial template switching activity, irrespective of its attachment site on PCNA. Our findings provide insight into the interrelationship between the two branches of damage bypass and suggest the existence of a yet unidentified, highly linkage-selective receptor of polyubiquitylated PCNA.

Structural basis of ubiquitin recognition by the winged-helix domain of Cockayne syndrome group B protein.

Cockayne syndrome group B (CSB, also known as ERCC6) protein is involved in many DNA repair processes and essential for transcription-coupled repair (TCR). The central region of CSB has the helicase motif, whereas the C-terminal region contains important regulatory elements for repair of UV- and oxidative stress-induced damages and double-strand breaks (DSBs). A previous study suggested that a small part (∼30 residues) within this region was responsible for binding to ubiquitin (Ub). Here, we show that the Ub-binding of CSB requires a larger part of CSB, which was previously identified as a winged-helix domain (WHD) and is involved in the recruitment of CSB to DSBs. We also present the crystal structure of CSB WHD in complex with Ub. CSB WHD folds as a single globular domain, defining a class of Ub-binding domains (UBDs) different from 23 UBD classes identified so far. The second α-helix and C-terminal extremity of CSB WHD interact with Ub. Together with structure-guided mutational analysis, we identified the residues critical for the binding to Ub. CSB mutants defective in the Ub binding reduced repair of UV-induced damage. This study supports the notion that DSB repair and TCR may be associated with the Ub-binding of CSB.

Expanding the type IIB DNA topoisomerase family: identification of new topoisomerase and topoisomerase-like proteins in mobile genetic elements.

The control of DNA topology by DNA topoisomerases is essential for virtually all DNA transactions in the cell. These enzymes, present in every organism, exist as several non-homologous families. We previously identified a small group of atypical type IIB topoisomerases, called Topo VIII, mainly encoded by plasmids. Here, taking advantage of the rapid expansion of sequence databases, we identified new putative Topo VIII homologs. Our analyses confirm the exclusivity of the corresponding genes to mobile genetic elements (MGE) and extend their distribution to nine different bacterial phyla and one archaeal superphylum. Notably, we discovered another subfamily of topoisomerases, dubbed 'Mini-A', including distant homologs of type IIB topoisomerases and encoded by extrachromosomal and integrated bacterial and archaeal viruses. Interestingly, a short, functionally uncharacterized motif at the C-terminal extremity of type IIB topoisomerases appears sufficient to discriminate between Mini-A, Topo VI and Topo VIII subfamilies. This motif could be a key element for understanding the differences between the three subfamilies. Collectively, this work leads to an updated model for the origin and evolution of the type IIB topoisomerase family and raises questions regarding the role of topoisomerases during replication of MGE in bacteria and archaea.

G-Quadruplexes in the Archaea Domain.

The importance of unusual DNA structures in the regulation of basic cellular processes is an emerging field of research. Amongst local non-B DNA structures, G-quadruplexes (G4s) have gained in popularity during the last decade, and their presence and functional relevance at the DNA and RNA level has been demonstrated in a number of viral, bacterial, and eukaryotic genomes, including humans. Here, we performed the first systematic search of G4-forming sequences in all archaeal genomes available in the NCBI database. In this article, we investigate the presence and locations of G-quadruplex forming sequences using the G4Hunter algorithm. G-quadruplex-prone sequences were identified in all archaeal species, with highly significant differences in frequency, from 0.037 to 15.31 potential quadruplex sequences per kb. While G4 forming sequences were extremely abundant in Hadesarchaea archeon (strikingly, more than 50% of the Hadesarchaea archaeon isolate WYZ-LMO6 genome is a potential part of a G4-motif), they were very rare in the Parvarchaeota phylum. The presence of G-quadruplex forming sequences does not follow a random distribution with an over-representation in non-coding RNA, suggesting possible roles for ncRNA regulation. These data illustrate the unique and non-random localization of G-quadruplexes in Archaea.

Structural insights into two distinct binding modules for Lys63-linked polyubiquitin chains in RNF168.

The E3 ubiquitin (Ub) ligase RNF168 plays a critical role in the initiation of the DNA damage response to double-strand breaks (DSBs). The recruitment of RNF168 by ubiquitylated targets involves two distinct regions, Ub-dependent DSB recruitment module (UDM) 1 and UDM2. Here we report the crystal structures of the complex between UDM1 and Lys63-linked diUb (K63-Ub2) and that between the C-terminally truncated UDM2 (UDM2ΔC) and K63-Ub2. In both structures, UDM1 and UDM2ΔC fold as a single α-helix. Their simultaneous bindings to the distal and proximal Ub moieties provide specificity for Lys63-linked Ub chains. Structural and biochemical analyses of UDM1 elucidate an Ub-binding mechanism between UDM1 and polyubiquitylated targets. Mutations of Ub-interacting residues in UDM2 prevent the accumulation of RNF168 to DSB sites in U2OS cells, whereas those in UDM1 have little effect, suggesting that the interaction of UDM2 with ubiquitylated and polyubiquitylated targets mainly contributes to the RNF168 recruitment.

Structural basis of the interaction between Topoisomerase IIIß and the TDRD3 auxiliary factor.

Topoisomerase IIIß (TOP3ß) is a DNA/RNA topoisomerase that has been implicated in epigenetic or translational control of gene expression. In cells, TOP3ß co-exists with its specific auxiliary factor, TDRD3. TDRD3 serves as a scaffold protein to recruit TOP3ß to its DNA/RNA substrates accumulating in specific cellular sites such as methylated chromatins or neural stress granules. Here we report the crystal structures of the catalytic domain of TOP3ß, the DUF1767-OB-fold domains of TDRD3 and their complex at 3.44 Å, 1.62 Å and 3.6 Å resolutions, respectively. The toroidal-shaped catalytic domain of TOP3ß binds the OB-fold domain of TDRD3. The TDRD3 OB-fold domain harbors the insertion loop, which is protruding from the core structure. Both the insertion loop and core region interact with TOP3ß. Our pull-down binding assays showed that hydrophobic characters of the core surface and the amino- and carboxy-terminal regions of the insertion loop are essential for the interaction. Furthermore, by comparison with the structure of the homologous Topoisomerase IIIα (TOP3α)-RMI1 complex, we identified Arg96, Val109, Phe139 and the short insertion loop of TDRD3 as the critical structural elements for the specific interaction with TOP3ß to avoid the non-cognate interaction with TOP3α.

Structural basis for ubiquitin recognition by ubiquitin-binding zinc finger of FAAP20.

Several ubiquitin-binding zinc fingers (UBZs) have been reported to preferentially bind K63-linked ubiquitin chains. In particular, the UBZ domain of FAAP20 (FAAP20-UBZ), a member of the Fanconi anemia core complex, seems to recognize K63-linked ubiquitin chains, in order to recruit the complex to DNA interstrand crosslinks and mediate DNA repair. By contrast, it is reported that the attachment of a single ubiquitin to Rev1, a translesion DNA polymerase, increases binding of Rev1 to FAAP20. To clarify the specificity of FAAP20-UBZ, we determined the crystal structure of FAAP20-UBZ in complex with K63-linked diubiquitin at 1.9 Å resolution. In this structure, FAAP20-UBZ interacts only with one of the two ubiquitin moieties. Consistently, binding assays using surface plasmon resonance spectrometry showed that FAAP20-UBZ binds ubiquitin and M1-, K48- and K63-linked diubiquitin chains with similar affinities. Residues in the vicinity of Ala168 within the α-helix and the C-terminal Trp180 interact with the canonical Ile44-centered hydrophobic patch of ubiquitin. Asp164 within the α-helix and the C-terminal loop mediate a hydrogen bond network, which reinforces ubiquitin-binding of FAAP20-UBZ. Mutations of the ubiquitin-interacting residues disrupted binding to ubiquitin in vitro and abolished the accumulation of FAAP20 to DNA damage sites in vivo. Finally, structural comparison among FAAP20-UBZ, WRNIP1-UBZ and RAD18-UBZ revealed distinct modes of ubiquitin binding. UBZ family proteins could be divided into at least three classes, according to their ubiquitin-binding modes.

Impaired calcium regulation of smooth muscle during chronic vasospasm following subarachnoid hemorrhage.

The intracellular calcium level was determined in the canine basilar artery to investigate whether Ca2+ regulation of its smooth muscle is altered during chronic vasospasm following subarachnoid hemorrhage. A double-hemorrhage model was used. The occurrence of vasospasm was confirmed angiographically 7 days after initial hemorrhage. The intracellular calcium concentration ([Ca2+]i) of smooth muscle was measured using Fura-2. Fluorescence to excitation at 340 and 356 nm was monitored and the ration R340/356 was used as the indicator of [Ca2+]i. When the extracellular calcium concentration ([Ca2+]e) was increased from pCa 8 to 2, [Ca2+]i also increased. In the spastic arteries, the [Ca2+]e - [Ca2+]i curve was elevated as compared with the normal arteries. Treatment with ionomycin elevated the curve in the normal group, but it had little effect in the spastic arteries. Values of [Ca2+]i, calculated in multiples of Kd, were greater in the spastic arteries. Diltiazem (10(-5) mol/L) partially suppressed the augmented [Ca2+]i signal in the spastic arteries, whereas it did not affect the curve in the control group. These results indicate that the calcium regulation of smooth muscle is impaired after subarachnoid hemorrhage, which may contribute to the pathogenesis of chronic vasospasm.

Twelve cases of jugular foramen neurinoma.

Twelve patients with jugular foramen neurinoma were operated on at our clinic between 1974 and 1990. The initial signs and symptoms were variable; dysfunctions of the 7th, 8th, 9th, 10th, and 11th nerves were frequently observed. Among these, involvement of the eighth nerve was most frequent, and three patients were given a misdiagnosis of acoustic neurinoma. Computed tomography scan and magnetic resonance imaging were useful not only for the correct diagnosis, but also for planning the surgical treatment and postoperative followup. Surgical resection was accomplished with four different approaches: (1) suboccipital approach without opening the jugular foramen, (2) suboccipital approach with opening the jugular foramen, (3) suboccipital approach with opening the jugular foramen combined with infralabyrinthine approach, (4) infralabyrinthine approach. The surgical approach depended on the presence of intracranial tumor and on the extent of extracranial involvement. There was no operative mortality. Dysfunction of the 8th, 9th, 10th, or 11th nerve did not improve in any patient after tumor removal. In contrast, 12th nerve palsy improved in two of three patients after tumor removal. The nerve of origin was identified in five cases; those were from the ninth nerve in three and from the 11th nerve in two.

[The development of radiosurgery--introduction of the gamma knife for brain lesions].

In 1990, the first gamma unit in Asia was installed at Tokyo University Hospital. This article describes the radiosurgical techniques used and clinical experiences during treatment of 70 cases at the unit. Stereotactic radiosurgery using a gamma knife for the treatment of AVM, CPA tumor and meningioma has been used for more than 20 years. With this form of therapy, a high dose of radiation is delivered from multi-directional beams to deep lesions of the brain, incurring only minimal radiation exposure at the periphery. When a large dose of radiation is applied to a small area, there may be certain side effects especially if care is not taken. Accordingly, treatment of brain lesions using a gamma knife requires teamwork, even though it appears to be a safe procedure at the present time.