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PubMed1993

Fusion of CHOP to a novel RNA-binding protein in human myxoid liposarcoma.

Human myxoid liposarcomas contain a characteristic chromosomal translocation, t(12;16)(q13;p11), that is associated with a structural rearrangement of the gene encoding CHOP, a growth arrest and DNA-damage inducible member of the C/EBP family of transcription factors residing on 12q13.1. Using a CHOP-specific complementary probe and antiserum we report here the presence of an abnormal CHOP transcript and protein in these tumours. Cloning of the translocation-associated CHOP gene product revealed a fusion between CHOP and a gene provisionally named TLS (translocated in liposarcoma). TLS is a novel nuclear RNA-binding protein with extensive sequence similarity to EWS, the product of a gene commonly translocated in Ewing's sarcoma. In TLS-CHOP the RNA-binding domain of TLS is replaced by the DNA-binding and leucine zipper dimerization domain of CHOP. Targeting of a conserved effector domain of RNA-binding proteins to DNA may play a role in tumour formation.

Amino Acid Sequence↗

PubMed2003

Evaluation of lateral resolution of scanning surface microscopy by total internal reflection with thermal lens effect.

We have developed a novel method for in situ and non-destructive surface analyses, or a total internal reflection with thermal lens spectroscopy (TIR-TLS), which has sufficient sensitivity to monitor phenomena in thin films, such as lipid bilayers. In this study, we applied TIR-TLS to microscopy for surface analyses, and we experimentally obtained its lateral resolution using the edge of a chromium film made by a photolithography technique. The obtained resolution was 20 microm, which was 60% of the diameter of an excitation beam at the interface. The estimated resolution with a simple model agreed with the experimental one, and from this model, TIR-TLS microscopy has the same resolution as that of ordinary optical microscopy. The microscopy by TIR-TLS was applied to a sample whose contrast was too weak to be visually seen, and an image of the sample was obtained without any loss of resolution.

Journal Article↗

PubMed2007

Accessory proteins assist exonuclease-deficient bacteriophage T4 DNA polymerase in replicating past an abasic site.

Replicative DNA polymerases, such as T4 polymerase, possess both elongation and 3'-5' exonuclease proofreading catalytic activities. They arrest at the base preceding DNA damage on the coding DNA strand and specialized DNA polymerases have evolved to replicate across the lesion by a process known as TLS (translesion DNA synthesis). TLS is considered to take place in two steps that often require different enzymes, insertion of a nucleotide opposite the damaged template base followed by extension from the inserted nucleotide. We and others have observed that inactivation of the 3'-5' exonuclease function of T4 polymerase enables TLS across a single site-specific abasic [AP (apurinic/apyrimidinic)] lesion. In the present study we report a role for auxiliary replicative factors in this reaction. When replication is performed with a large excess of DNA template over DNA polymerase in the absence of auxiliary factors, the exo- polymerase (T4 DNA polymerase deficient in the 3'-5' exonuclease activity) inserts one nucleotide opposite the AP site but does not extend past the lesion. Addition of the clamp processivity factor and the clamp loader complex restores primer extension across an AP lesion on a circular AP-containing DNA substrate by the exo- polymerase, but has no effect on the wild-type enzyme. Hence T4 DNA polymerase exhibits a variety of responses to DNA damage. It can behave as a replicative polymerase or (in the absence of proofreading activity) as a specialized DNA polymerase and carry out TLS. As a specialized polymerase it can function either as an inserter or (with the help of accessory proteins) as an extender. The capacity to separate these distinct functions in a single DNA polymerase provides insight into the biochemical requirements for translesion DNA synthesis.

Bacteriophage T4↗

PubMed1999

Acute tumour lysis syndrome: a case in AL amyloidosis.

Tumour lysis syndrome (TLS) in plasma cell dyscrasias is extremely rare. TLS has been described in eight cases of multiple myeloma undergoing high-dose therapy with autologous stem cell transplant (ASCT). Recently, clinical trials of intensive chemotherapy followed by autologous or allogeneic stem cell support has been shown to offer potential benefit in AL (amyloid light-chain) amyloidosis. TLS in primary AL amyloidosis in this setting has not been previously reported. We report a case of TLS in a patient with AL amyloidosis which developed after high-dose melphalan chemotherapy supported by ASCT.

Acute Disease↗

PubMed2001

Roles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coli.

DNA polymerase V, composed of a heterotrimer of the DNA damage-inducible UmuC and UmuD(2)(') proteins, working in conjunction with RecA, single-stranded DNA (ssDNA)-binding protein (SSB), beta sliding clamp, and gamma clamp loading complex, are responsible for most SOS lesion-targeted mutations in Escherichia coli, by catalyzing translesion synthesis (TLS). DNA polymerase II, the product of the damage-inducible polB (dinA ) gene plays a pivotal role in replication-restart, a process that bypasses DNA damage in an error-free manner. Replication-restart takes place almost immediately after the DNA is damaged (approximately 2 min post-UV irradiation), whereas TLS occurs after pol V is induced approximately 50 min later. We discuss recent data for pol V-catalyzed TLS and pol II-catalyzed replication-restart. Specific roles during TLS for pol V and each of its accessory factors have been recently determined. Although the precise molecular mechanism of pol II-dependent replication-restart remains to be elucidated, it has recently been shown to operate in conjunction with RecFOR and PriA proteins.

Catalysis↗

PubMed2005

The 9-1-1 checkpoint clamp physically interacts with polzeta and is partially required for spontaneous polzeta-dependent mutagenesis in Saccharomyces cerevisiae.

The use of translesion synthesis (TLS) polymerases to bypass DNA lesions during replication constitutes an important mechanism to restart blocked/stalled DNA replication forks. Because TLS polymerases generally have low fidelity on undamaged DNA, the cell must regulate the interaction of TLS polymerases with damaged versus undamaged DNA to maintain genome integrity. The Saccharomyces cerevisiae checkpoint proteins Ddc1, Rad17, and Mec3 form a clamp-like structure (the 9-1-1 clamp) that has physical similarity to the homotrimeric sliding clamp proliferating cell nuclear antigen, which interacts with and promotes the processivity of the replicative DNA polymerases. In this work, we demonstrate both an in vivo and in vitro physical interaction between the Mec3 and Ddc1 subunits of the 9-1-1 clamp and the Rev7 subunit of the Polzeta TLS polymerase. In addition, we demonstrate that loss of Mec3, Ddc1, or Rad17 results in a decrease in Polzeta-dependent spontaneous mutagenesis. These results suggest that, in addition to its checkpoint signaling role, the 9-1-1 clamp may physically regulate Polzeta-dependent mutagenesis by controlling the access of Polzeta to damaged DNA.

Base Sequence↗

PubMed2003

A longitudinal approach for constructing beta-expectation tolerance intervals.

A new method is presented for constructing beta-expectation tolerance limits (TLs) for longitudinal data with error components regression structure. The TLs are mainly intended for small samples where the variation between and within subjects is large. In such cases the new method is superior to existing methods, which are based on a cross-sectional approach and which do not utilize the longitudinal structure of the data. Simulation studies show that the mean length of the TLs can be much reduced by using the new approach, while at the same time the beta-expectation property is maintained. The gain from using the longitudinal approach furthermore increases with the beta-expectation level. The results are demonstrated on data consisting of measurements of glutamate concentrations in brains from rats. Here, the cross-sectional approach due to Wilks give 90-99% TLs, which are up to 101-230% wider than are those obtained with the new approach.

Animals↗

PubMed2005

Laparoscopic splenectomy for hematologic disorders: experience with the first fifty patients.

BACKGROUND: Splenectomy is increasingly being performed by various minimal access surgical modalities for select hematologic disorders. METHODS: A retrospective analysis was performed on the first 50 patients on whom laparoscopic splenectomy (LS) was attempted. The data studied included indications for surgery, patient demographics, intraoperative parameters, and patient outcomes. A total laparoscopic approach (TLS) was employed in 38 patients and a hand-assisted technique (HALS) was used in 12 patients with massive splenomegaly. Eight patients had concomitant surgical procedures: 7 patients underwent laparoscopic cholecystectomy and 1 patient received a kidney transplant. The most common indications for LS were idiopathic thrombocytopenic purpura (ITP) (50%) and hereditary spherocytosis (24%). RESULTS: LS was successfully completed in 48 patients (96%). Thirty-four patients (68%) required perioperative blood or platelet transfusions. The mean spleen diameter was 17.1 cm (range, 11.2-28.4 cm) on imaging study and mean intact splenic weight was 1019 gm. The mean operative time was 188 minutes (range, 90-340 minutes) in the TLS group and 171 minutes (range, 120-240 minutes) in the HALS group. The mean intraoperative blood loss was 306 mL (range, 40-640 mL) in the TLS group and 163 mL (range, 100-300 mL) in the HALS group. The mean postoperative hospital stay was 3.2 days (range, 2-5 days). CONCLUSION: TLS is safe and feasible in patients with nonpalpable spleens. A concomitant laparoscopic procedure for treating coexisting abdominal pathology may be performed without additional morbidity. The HALS technique may be preferable in patients with splenomegaly (palpable spleens), as it appears to offer intraoperative advantages for retraction, dissection, hemostasis, and organ retrieval.

Blood Loss, Surgical↗

PubMed2002

RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells.

Translesion DNA synthesis (TLS) and homologous DNA recombination (HR) are two major pathways that account for survival after post-replicational DNA damage. TLS functions by filling gaps on a daughter strand that remain after DNA replication caused by damage on the mother strand, while HR can repair gaps and breaks using the intact sister chromatid as a template. The RAD18 gene, which is conserved from lower eukaryotes to vertebrates, is essential for TLS in Saccharomyces cerevisiae. To investigate the role of RAD18, we disrupted RAD18 by gene targeting in the chicken B-lymphocyte line DT40. RAD18(-/-) cells are sensitive to various DNA-damaging agents including ultraviolet light and the cross-linking agent cisplatin, consistent with its role in TLS. Interestingly, elevated sister chromatid exchange, which reflects HR- mediated post-replicational repair, was observed in RAD18(-/-) cells during the cell cycle. Strikingly, double mutants of RAD18 and RAD54, a gene involved in HR, are synthetic lethal, although the single mutant in either gene can proliferate with nearly normal kinetics. These data suggest that RAD18 plays an essential role in maintaining chromosomal DNA in cooperation with the RAD54-dependent DNA repair pathway.

Amino Acid Sequence↗

PubMed2003

Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis.

Pol kappa and Rev1 are members of the Y family of DNA polymerases involved in tolerance to DNA damage by replicative bypass [translesion DNA synthesis (TLS)]. We demonstrate that mouse Rev1 protein physically associates with Pol kappa. We show too that Rev1 interacts independently with Rev7 (a subunit of a TLS polymerase, Pol zeta) and with two other Y-family polymerases, Pol iota and Pol eta. Mouse Pol kappa, Rev7, Pol iota and Pol eta each bind to the same approximately 100 amino acid C-terminal region of Rev1. Furthermore, Rev7 competes directly with Pol kappa for binding to the Rev1 C-terminus. Notwithstanding the physical interaction between Rev1 and Pol kappa, the DNA polymerase activity of each measured by primer extension in vitro is unaffected by the complex, either when extending normal primer-termini, when bypassing a single thymine glycol lesion, or when extending certain mismatched primer termini. Our observations suggest that Rev1 plays a role(s) in mediating protein-protein interactions among DNA polymerases required for TLS. The precise function(s) of these interactions during TLS remains to be determined.

Animals↗

PubMed2005

The BRCT domain of mammalian Rev1 is involved in regulating DNA translesion synthesis.

Rev1 is a deoxycytidyl transferase associated with DNA translesion synthesis (TLS). In addition to its catalytic domain, Rev1 possesses a so-called BRCA1 C-terminal (BRCT) domain. Here, we describe cells and mice containing a targeted deletion of this domain. Rev1(B/B) mice are healthy, fertile and display normal somatic hypermutation. Rev1(B/B) cells display an elevated spontaneous frequency of intragenic deletions at Hprt. In addition, these cells were sensitized to exogenous DNA damages. Ultraviolet-C (UV-C) light induced a delayed progression through late S and G2 phases of the cell cycle and many chromatid aberrations, specifically in a subset of mutant cells, but not enhanced sister chromatid exchanges (SCE). UV-C-induced mutagenesis was reduced and mutations at thymidine-thymidine dimers were absent in Rev1(B/B) cells, the opposite phenotype of UV-C-exposed cells from XP-V patients, lacking TLS polymerase eta. This suggests that the enhanced UV-induced mutagenesis in XP-V patients may depend on error-prone Rev1-dependent TLS. Together, these data indicate a regulatory role of the Rev1 BRCT domain in TLS of a limited spectrum of endogenous and exogenous nucleotide damages during a defined phase of the cell cycle.

Animals↗

PubMed2002

Large-bandgap behavior in transport of electrons through individual DNA molecules caused by coupling with a two-level system.

We propose a model to interpret the large-bandgap behavior in transport of electrons through an individual DNA molecule where the tunneling electrons are coupled with two-level systems (TLS). The TLS can be regarded as the simplest way to describe vibrations and inelastic scattering in the molecules if the two levels represent the low-lying phonon states. The nonlinear current-voltage curves can be derived by the use of the transfer matrices in an equivalent single-particle multichannel network. At low temperatures, the gap of the conduction band is sensitive to the strength of the coupling between the TLS and the conduction electrons. It is shown that the large-bandgap behavior similar to that of semiconductors stems from the inelastic scattering by the TLS.

Biophysics↗

PubMed2005

Tunneling spectroscopy of two-level systems inside a Josephson junction.

We consider a two-level system (TLS) with energy level separation plankvOmega0 inside a Josephson junction. The junction is shunted by a resistor R and is voltage V biased. If the TLS modulates the Josephson energy and/or is optically active, it is Rabi driven by the Josephson oscillations in the running phase regime near the resonance 2eV=plankvOmega0. The Rabi oscillations, in turn, translate into oscillations of current and voltage that can be detected in noise measurements. This effect provides an option to fully characterize the TLS inside Josephson junction and to find the TLS's contribution to the decoherence when the junction is used as a qubit.

Journal Article↗

PubMed2005

What is moving in silica at 1 K? A computer study of the low-temperature anomalies.

Though the existence of two-level systems (TLS) is widely accepted to explain low-temperature anomalies in many physical observables, knowledge about their properties is very rare. For silica, which is one of the prototype glass-forming systems, we elucidate the properties of the TLS via computer simulations by applying a systematic search algorithm. We get specific information in the configuration space, i.e., about relevant energy scales, the absolute number of TLS, and electric dipole moments. Furthermore, important insight about the real-space realization of the TLS can be obtained. Comparison with experimental observations is included.

Journal Article↗

PubMed2006

Analyses of ultraviolet-induced focus formation of hREV1 protein.

Translesional DNA synthesis (TLS) is one of the DNA damage tolerance mechanisms that allow cells with DNA damage to continue DNA replication. Each of the mammalian Y-family DNA polymerases (Pol eta, Pol iota, Pol kappa, and REV1) has been shown to carry out TLS by itself or in combination with another enzyme in vitro. Recently, the C-terminal region of mammalian REV1 (the total 1251 residues in human) was found to interact with Pol eta, Pol iota, and Pol kappa, as well as with the REV7 subunit of another TLS enzyme, Pol zeta. Thus, it is proposed that REV1 plays a pivotal role in TLS in vivo. We here describe our study on the localization of human REV1 protein (hREV1) in nondamaged and ultraviolet (UV)-irradiated cells. Ectopically expressed hREV1 in mammalian cells was localized to the nucleus and exhibited dozens of tiny foci in approximately 3% of nondamaged cells. The percentage of focus-forming cells markedly increased after UV irradiation in a time- and dose-dependent manner. The focus formation was associated with UV-induced DNA damage. Interestingly, although the hREV1 foci in S-phase cells colocalized with PCNA foci, suggesting the association of hREV1 with the replication machinery, hREV1 focus formation was observed not only in the S phase but also outside S phase. Furthermore, it was found that the hREV1 focus formation after UV irradiation required a region near the C-terminal (826-1178).

Animals↗

PubMed2005

Expression and possible functions of DNA lesion bypass proteins in spermatogenesis.

In mammalian cells, there is a complex interplay of different DNA damage response and repair mechanisms. Several observations suggest that, in particular in gametogenesis, proteins involved in DNA repair play an intricate role in and outside the context of DNA repair. Here, we discuss the possible roles of proteins that take part in replicative damage bypass (RDB) mechanisms, also known as post-replication DNA repair (PRR), in germ line development. In yeast, and probably also in mammalian somatic cells, RDB [two subpathways: damage avoidance and translesion synthesis (TLS)] prevents cessation of replication forks during the S phase of the cell cycle, in situations when the replication machinery encounters a lesion present in the template DNA. Many genes encoding proteins involved in RDB show an increased expression in testis, in particular in meiotic and post-meiotic spermatogenic cells. Several RDB proteins take part in protein ubiquitination, and we address relevant aspects of the ubiquitin system in spermatogenesis. RDB proteins might be required for damage avoidance and TLS of spontaneous DNA damage during gametogenesis. In addition, we consider the possible functional relation between TLS and the induction of mutations in spermatogenesis. TLS requires the activity of highly specialized polymerases, and is an error-prone process that may induce mutations. In evolutionary terms, controlled generation of a limited number of mutations in gametogenesis might provide a mechanism for evolvability.

Animals↗

PubMed2005

Mutant forms of the Escherichia colibeta sliding clamp that distinguish between its roles in replication and DNA polymerase V-dependent translesion DNA synthesis.

The Escherichia colibeta sliding clamp is proposed to play an important role in regulating DNA polymerase traffic at the replication fork. As part of an ongoing effort to understand how organisms manage the actions of their multiple DNA polymerases, we examined the ability of several mutant forms of the beta clamp to function in DNA polymerase V- (pol V-) dependent translesion DNA synthesis (TLS) in vivo. Our results indicate that a dnaN159 strain, which expresses a temperature sensitive form of the beta clamp, was impaired for pol V-dependent TLS at the permissive temperature of 37 degrees C. This defect was complemented by a plasmid that expressed near-physiological levels of the wild-type clamp. Using a dnaN159 mutant strain, together with various plasmids expressing mutant forms of the clamp, we determined that residues H148 through R152, which comprise a portion of a solvent exposed loop, as well as position P363, which is located in the C-terminal tail of the beta clamp, are critically important for pol V-dependent TLS in vivo. In contrast, these same residues appear to be less critical for pol III-dependent replication. Taken together, these findings indicate that: (i) the beta clamp plays an essential role in pol V-dependent TLS in vivo and (ii) pol III and pol V interact with non-identical surfaces of the beta clamp.

Amino Acid Sequence↗

PubMed2025

Enzymatic and Structural Roles of Candida albicans Rev1 in DNA Damage Response and Disseminated Candidiasis.

Translesion DNA synthesis (TLS) is a fundamental biological process that enables DNA replication through various lesions to ensure genome stability and to prevent cell death due to replication fork collapse. Rev1, a member of Y-family DNA polymerase (Pol), functions in concert with a B-family enzyme Polζ in promoting TLS through various lesions. Interestingly, for such a function, the catalytic activity of Rev1 seems to be dispensable in Saccharomyces cerevisiae. Unlike Polζ, which possesses robust DNA polymerase activity, biochemical assays suggest that Rev1 predominantly incorporates a "C" opposite any templating residues, but the biological relevance of this activity of Rev1 remains elusive. Here we characterized Rev1 from Candida albicans, an opportunistic fungal pathogen responsible for maximum casualties due to systemic candidiasis in immunosuppressed individuals. Concerted genetic analyses of several Rev1 mutants in various DNA-damaging conditions suggested that in most lesion bypasses except 4-NQO-induced DNA lesions, the catalytic role of Rev1 is not important. However, simultaneous interactions of BRCT and the C-terminal domain of Rev1 with PCNA and Polζ, respectively, enable Rev1 to be essential during TLS. DNA damage recovery and mutagenesis assays further confirmed the lesion-specific roles of various domains of Rev1. Contrary to ex vivo data, animal studies suggested that CaRev1 is dispensable for systemic candidiasis development. We discuss the possible involvement of other TLS DNA polymerases in DNA damage response while C. albicans replicates and establishes itself in the host.

Candida albicans↗