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PubMed2006

ATR homolog Mec1 controls association of DNA polymerase zeta-Rev1 complex with regions near a double-strand break.

DNA polymerase zeta (Polzeta) and Rev1 contribute to the bypassing of DNA lesions, termed translesion DNA synthesis (TLS). Polzeta consists of two subunits, one encoded by REV3 (the catalytic subunit) and the other encoded by REV7. Rev1 acts as a deoxycytidyl transferase, inserting dCMP opposite lesions. Polzeta and Rev1 have been shown to operate in the same TLS pathway in the budding yeast Saccharomyces cerevisiae. Here, we show that budding yeast Polzeta and Rev1 form a complex and associate together with double-strand breaks (DSBs). As a component of the Polzeta-Rev1 complex, Rev1 plays a noncatalytic role in the association with DSBs. In budding yeast, the ATR-homolog Mec1 plays a central role in the DNA-damage checkpoint response. We further show that Mec1-dependent phosphorylation promotes the Polzeta-Rev1 association with DSBs. Rev1 association with DSBs requires neither the function of the Rad24 checkpoint-clamp loader nor the Rad6-Rad18-mediated ubiquitination of PCNA. Our results reveal a novel role of Mec1 in the localization of the Polzeta-Rev1 complex to DNA lesions and highlight a linkage of TLS polymerases to the checkpoint response.

DNA Damage↗

PubMed2004

Polymerase zeta dependency of increased adaptive mutation frequencies in nucleotide excision repair-deficient yeast strains.

Reversions of an auxotrophy-causing frameshift allele during prolonged starvation of yeast cells were used as a means to elucidate the mechanisms concerned with the generation of spontaneous adaptive mutations in cell cycle-arrested cells. Whereas about 50% of these reversions were previously shown to depend on the non-homologous end joining pathway of DNA double-strand break repair, the origin of the residual 50% remains unknown. In search for a mechanism for generation of the latter fraction of reversions we examined the role of the translesion synthesis (TLS) polymerases zeta, eta and Rev1p in cells with wild-type or impaired nucleotide excision repair (NER) capacity. The basal level of adaptive mutations in the repair-proficient wild type was not influenced by disruptions of the genes coding for these three TLS polymerases. Intriguingly, a deficiency in NER by disruption of RAD14, RAD16 or RAD26 resulted in a significantly higher frequency of adaptive mutation, yet this increase was strictly dependent on an intact REV3 gene, coding for the catalytic subunit of polymerase zeta. Furthermore, we observed that intact REV3 was also required for the occurrence of increased frequencies of adaptive mutants in the NER-proficient wild type following UV irradiation. While in proliferating cells the translesion synthesis function of polymerase zeta is connected to DNA replication, our data suggest that in cell cycle-arrested cells this enzyme is able to carry out either TLS or error-prone polymerization along an undamaged template in the course of repair processes. Such a hitherto unappreciated activity of polymerase zeta in non-replicating cells may contribute to the incidence of mutations in evolution, aging and cancer.

Base Sequence↗

PubMed2004

TTF-1 expression is specific for lung primary in typical and atypical carcinoids: TTF-1-positive carcinoids are predominantly in peripheral location.

Thyroid transcription factor (TTF)-1 expression in neuroendocrine tumors (NETs) has not been studied as widely as that in non-NETs, with the exception of small cell carcinomas, in which TTF-1 is highly sensitive but not specific for a primary lung tumor. The reported incidence of TTF-1 expression in pulmonary carcinoids has also been highly variable in the literature. To evaluate the expression of TTF-1 in NETs and potential value of TTF-1 in distinguishing pulmonary NETs from those of extrapulmonary origin, we performed an immunohistochemical study by using semiquantitative analysis on formalin-fixed, paraffin-embedded sections from 111 NETs, including 80 pulmonary (11 carcinoid tumorlets [TLs] or foci of neuroendocrine cell hyperplasia [NEH], 36 typical carcinoids [TCs], 17 atypical carcinoids [ACs], 16 large cell neuroendocrine carcinomas [LCNECs]), 13 thymic (3 TCs, 8 ACs, 2 LCNECs), 17 gastrointestinal or pancreatic (13 TCs, 4 ACs), and 1 ovarian (LCNEC). Pulmonary carcinoids were subdivided into those with central and those with peripheral location. TTF-1 positivity was seen exclusively in pulmonary NETs and was significantly higher in NEH or TLs (72.7%) than in TCs (27.8%), ACs (29.4%), and LCNECs (37.5%; P = 0.03). All extrapulmonary NETs were uniformly negative for TTF-1 staining. Interestingly, 12 of 14 TTF-1-positive pulmonary TCs and ACs had a peripheral location with spindle cell morphology, as did all cases of TL, a purported precursor of peripheral carcinoids. In conclusion, TTF-1 expression was 100% specific, though not so sensitive, for the lung primary in TCs and ACs and possibly also in LCNECs. Prevalent TTF-1 positivity in TLs and peripheral carcinoids suggest that they may be histogenetically distinct from the central carcinoids, which are typically composed of TTF-1-negative, more rounded cells.

Aged↗

PubMed2005

An update on the role of translesion synthesis DNA polymerases in Ig hypermutation.

Several years have passed since the discovery of activation-induced cytosine deaminase (AID), the molecule responsible for triggering hypermutation of Ig genes. We now know that AID deaminates cytosines in the DNA encoding the variable portion of the Ig receptor, although an additional role in deaminating a regulatory mRNA transcript has not been ruled out. A major question that remains unanswered is how AID, a cytosine deaminase, causes mutations at both G:C and A:T base pairs. Mounting evidence suggests the involvement of a group of error-prone DNA polymerases known to bypass DNA lesions: the translesion synthesis (TLS) DNA polymerases. In this Review, we discuss the evidence for a role of TLS DNA polymerases in Ig hypermutation and argue that a major remaining challenge in our understanding of this mechanism is the recruitment of TLS DNA polymerases to the Ig locus following AID-mediated cytosine deamination.

Animals↗

PubMed2006

DNA repair in antibody somatic hypermutation.

Somatic hypermutation (SHM) underlies the generation of a diverse repertoire of high-affinity antibodies. It is effected by a two-step process: (i) DNA lesions initiated by activation-induced cytidine deaminase (AID), and (ii) lesion repair by the combined intervention of DNA replication and repair factors that include mismatch repair (MMR) proteins and translesion DNA synthesis (TLS) polymerases. AID and TLS polymerases that are crucial to SHM, namely polymerase (pol) theta, pol zeta and pol eta, are induced in B cells by the stimuli that are required to trigger this process: B-cell receptor crosslinking and CD40 engagement by CD154. These polymerases, together with MMR proteins and other DNA replication and repair factors, could assemble to form a multimolecular complex ("mutasome") at the site of DNA lesions. Molecular interactions in the mutasome would result in a "polymerase switch", that is, the substitution of the high-fidelity replicative pol delta and pol epsilon with the TLS pol theta, pol eta, Rev1, pol zeta and, perhaps, pol iota, which are error-prone and crucially insert mismatches or mutations while repairing DNA lesions. Here, we place these concepts in the context of the existing in vivo and in vitro findings, and discuss an integrated mechanistic model of SHM.

Animals↗

PubMed2006

Site-directed mutagenesis in the fingers subdomain of HIV-1 reverse transcriptase reveals a specific role for the beta3-beta4 hairpin loop in dNTP selection.

HIV-1 reverse transcriptase shares the key features of high fidelity polymerases, such as a closed architecture of the active site, but displays a level of fidelity that is intermediate to that of high fidelity, replicative polymerases and low fidelity translesion synthesis (TLS) polymerases. The beta3-beta4 loop of the HIV-1 RT fingers subdomain makes transient contacts with the dNTP and template base. To investigate the role of active site architecture in HIV-1 RT fidelity, we truncated the beta3-beta4 loop, eliminating contact between Lys65 and the gamma-phosphate of dNTP. The mutant, in a manner reminiscent of TLS polymerases, was only able to incorporate a nucleotide that was capable of base-pairing with the template nucleotide, but not a nucleotide shape-analog incapable of Watson-Crick hydrogen bonding. Unexpectedly, however, the deletion mutant differed from the TLS polymerases in that it displayed an increased fidelity. The increased fidelity was associated with reduced dNTP binding affinity as measured using the dead end complex formation. In an effort to delineate the specific amino acid residue in the deleted segment responsible for this phenotype, we examined the K65 residue. Two substitution mutants, K65R and K65A were studied. The K65A mutant behaved similarly to the deletion mutant displaying dependence on Watson-Crick hydrogen bonding, increased fidelity and reduced dNTP-binding, while the K65R was more akin to wild-type enzyme. These results underscore the key role of the K65 residue in the phenotype observed in the deletion mutant. Based on the well-known electrostatic interaction between K65 and the gamma-phosphate moiety of incoming dNTP substrate in the ternary complex structure of HIV-1 RT, we conclude that non-discriminatory interactions between beta3-beta4 loop and the dNTP in wild-type HIV-1 RT help lower dNTP selectivity. Our results show that the fidelity of dNTP insertion is influenced by protein interactions with the triphosphate moiety.

Alanine↗

PubMed2006

Closed-loop nonlinear system identification via the vector optimal parameter search algorithm: application to heart rate baroreflex control.

The vector optimal parameter search (VOPS) and the constrained optimal parameter search (COPS) are recently developed algorithms for closed-loop linear system identification. We extend both algorithms to be applicable to a closed-loop nonlinear system, which is characterized by a vector nonlinear autoregressive model. Monte Carlo simulations of nonlinear closed-loop systems were performed to compare the performance of the VOPS to the widely utilized vector least squares (VLS), the COPS and the total least squares (TLS) approaches. The relative error and linear transfer functions are computed to determine the accuracy of each method. The comparative results show that both the VOPS and COPS algorithms provide far superior parameter estimates than does the VLS for all simulation examples considered. The TLS provides better estimates than the VOPS, COPS and VLS when there is only observation noise present in the data. However, the performance of the TLS degrades considerably when the data are corrupted by dynamic noise. The clinical applicability of the two extended methods is examined by applying them to a classical physiological closed-loop system, the heart rate baroreflex. It was found that while both control and blockade of parasympathetic system conditions are dominated by linear dynamics, more nonlinearity was observed in the latter. This observation is statistically supported by the calculation of the mutual information of the data and their surrogates.

Algorithms↗

PubMed2026

Mature Tertiary Lymphoid Structures in Breast Cancers Are Associated With Antitumor Immunity and Better Prognosis.

Tertiary lymphoid structures (TLSs) are immune cells accumulated in nonlymphoid tissues, with an inner core of B cells encompassed by T cells. The aim of this study was to evaluate the clinical importance of mature TLSs in breast cancer, including their association with immunotherapy response and their role in modulating the tumor immune microenvironment. We analyzed histopathological data of 726 consecutive primary breast cancers and transcriptomic data of 824 breast cancer samples from the publicly available The Cancer Genome Atlas database to estimate the clinical and immunological values of mature TLSs in breast cancer. Additionally, we utilized pretreatment transcriptomic data of 69 patients with breast cancer from the publicly available I-SPY2 clinical trial to investigate the relation between TLS-related gene signatures and patient responses to immune checkpoint inhibitors. The existence of mature TLSs was identified in ⁓5.6% (41/726) of all patients with breast cancer (hormone receptor-positive human epidermal growth factor receptor-2 negative (HR+HER2-): 0.92%; triple-negative breast cancer (TNBC): 14.96%; and human epidermal growth factor receptor-2 positive (HER2+): 10.98%) and was independently associated with improved recurrence-free survival after adjusting for subtypes, tumor-infiltrating lymphocyte levels, and tumor stage after the multivariable Cox regression analysis in our patient cohort. Notably, the presence of mature TLSs was related to immune cell infiltration in our breast cancer patient cohort. In line with these findings, TLS-related gene signatures analyzed through transcriptomic data reliably reflected the existence of mature TLSs and were related to better clinical responses to immune checkpoint inhibitors in patients with breast cancer. In conclusion, our findings show that mature TLS formation is linked with immune cell infiltration, contributes to a favorable prognosis, and may function as a potential complementary biomarker for immunotherapy response in breast cancer.

Humans↗

PubMed2005

DNA polymerase V and RecA protein, a minimal mutasome.

A hallmark of the Escherichia coli SOS response is the large increase in mutations caused by translesion synthesis (TLS). TLS requires DNA polymerase V (UmuD'2C) and RecA. Here, we show that pol V and RecA interact by two distinct mechanisms. First, pol V binds to RecA in the absence of DNA and ATP and second, through its UmuD' subunit, requiring DNA and ATP without ATP hydrolysis. TLS occurs in the absence of a RecA nucleoprotein filament but is inhibited in its presence. Therefore, a RecA nucleoprotein filament is unlikely to be required for SOS mutagenesis. Pol V activity is severely diminished in the absence of RecA or in the presence of RecA1730, a mutant defective for pol V mutagenesis in vivo. Pol V activity is strongly enhanced with RecA mutants constitutive for mutagenesis in vivo, suggesting that RecA is an obligate accessory factor that activates pol V for SOS mutagenesis.

Adenosine Triphosphate↗

PubMed2005

Trading places: how do DNA polymerases switch during translesion DNA synthesis?

The replicative bypass of base damage in DNA (translesion DNA synthesis [TLS]) is a ubiquitous mechanism for relieving arrested DNA replication. The process requires multiple polymerase switching events during which the high-fidelity DNA polymerase in the replication machinery arrested at the primer terminus is replaced by one or more polymerases that are specialized for TLS. When replicative bypass is fully completed, the primer terminus is once again occupied by high-fidelity polymerases in the replicative machinery. This review addresses recent advances in our understanding of DNA polymerase switching during TLS in bacteria such as E. coli and in lower and higher eukaryotes.

Bacterial Proteins↗

PubMed2004

Detection of Bacillus endospores using total luminescence spectroscopy.

Detection and analysis of bacteria from environmental samples (e.g. water, air, and food) are usually accomplished by standard culture techniques or by analyses that target specific DNA sequences, antigens or chemicals. For large cell numbers in aqueous suspensions, an alternative technique that has proven useful is total luminescence spectroscopy (TLS). TLS is the acquisition of fluorescence data that records the unique excitation-emission matrix (EEM) of compound fluorophores. Past work has shown that one type of bacterial endospore, Bacillus megaterium, possessed a distinct EEM pattern useful for differentiating it in complex biological fluids and suspensions. The work described here extends those observations to establish some limits on the sensitivity and specificity of TLS for the detection and analysis of bacterial endospores versus (bacterial) vegetative cells in aqueous culture. Our findings show Bacillus endospores exhibit a dramatic blue shift of 130 nm in excitation and a smaller shift of 50 nm in emission when compared to ancillary endospore and non-endospore forming bacterial cells.

Bacillus↗

PubMed2004

The tRNA-like structure of Turnip yellow mosaic virus RNA is a 3'-translational enhancer.

Many positive stand RNA viral genomes lack the poly(A) tail that is characteristic of cellular mRNAs and that promotes translation in cis. The 3' untranslated regions (UTRs) of such genomes are expected to provide similar translation-enhancing properties as a poly(A) tail, yet the great variety of 3' sequences suggests that this is accomplished in a range of ways. We have identified a translational enhancer present in the 3' UTR of Turnip yellow mosaic virus (TYMV) RNA using luciferase reporter RNAs with generic 5' sequences transfected into plant cells. The 3' terminal 109 nucleotides comprising the tRNA-like structure (TLS) and an upstream pseudoknot (UPSK) act in synergy with a 5'-cap to enhance translation, with a minor contribution in stabilizing the RNA. Maximum enhancement requires that the RNA be capable of aminoacylation, but either the native valine or engineered methionine is acceptable. Mutations that decrease the affinity for translation elongation factor eEF1A (but also diminish aminoacylation efficiency) strongly decrease translational enhancement, suggesting that eEF1A is mechanistically involved. The UPSK seems to act as an important, though nonspecific, spacer element ensuring proper presentation of a functional TLS. Our studies have uncovered a novel type of translational enhancer and a new role for a plant viral TLS.

3' Untranslated Regions↗

PubMed2005

Replication-independent expression of genome components and capsid protein of brome mosaic virus in planta: a functional role for viral replicase in RNA packaging.

To begin elucidation of the relationship between Brome mosaic virus (BMV) replication and encapsidation, we used a T-DNA-based Agrobacterium-mediated transient expression (agroinfiltration) system in Nicotiana benthamiana leaves to express either individual or desired pairs of the three genomic RNAs. The packaging competence of these RNAs into virions formed by the transiently expressed coat protein (CP) was analyzed. We found that in the absence of a functional replicase, assembled virions contained non-replicating viral RNAs (RNA1 or RNA2 or RNA3 or RNA1 + RNA3 or RNA2 + RNA3) as well as cellular RNAs. By contrast, virions assembled in the presence of a functional replicase contained only viral RNAs. To further elucidate the specificity exhibited by the functional viral replicase in RNA packaging, replication-defective RNA1 and RNA2 were constructed by deleting the 3' tRNA-like structure (3' TLS). Co-expression of TLS-less RNA1 and RNA2 with wt RNA3 resulted in efficient synthesis of subgenomic RNA4. Virions recovered from leaves co-expressing TLS-less RNA1 and RNA2 and either CP mRNA or wt RNA3 exclusively contained viral RNAs. These results demonstrated that packaging of BMV genomic RNAs is not replication dependent whereas expression of a functional viral replicase plays an active role in increasing specificity of RNA packaging.

Base Sequence↗

PubMed2001

Blunt abdominal aortic trauma in association with thoracolumbar spine fractures.

All patients with blunt abdominal aortic disruption (BAAD) in the trauma registries at the three Regional Trauma Centres were retrospectively reviewed over the last decade. From the 11465 trauma admissions ISS>16,194 sustained aortic injuries. Eight cases of BAAD were identified, six with concurrent thoracolumbar spine (TLS) fractures (mean ISS 42). Patients with BAAD and TLS were subject to a detailed analysis. Clinically, three injury types were seen, hemodynamically unstable (uncontained full thickness laceration), stable symptomatic (intimal dissection with occlusion), and stable asymptomatic (contained full thickness laceration or intimal dissection without occlusion). All spinal column fractures involved a distractive mechanism, one with both distractive and translational fracture components. We propose that a distractive force, applied to the aorta lying anterior to the anterior longitudinal ligament, results in an aortic injury spectrum ranging from an intimal tear to a full thickness laceration, as a related injury. Computed tomography (CT) was an important imaging modality in the stable asymptomatic patients. All intimal dissections without occlusion were managed non-operatively. With distractive TLS fractures, BAAD needs to be considered.

Abdominal Injuries↗

PubMed2003

The structure and thermal motion of the B800-850 LH2 complex from Rps.acidophila at 2.0A resolution and 100K: new structural features and functionally relevant motions.

The structure at 100K of integral membrane light-harvesting complex II (LH2) from Rhodopseudomonas acidophila strain 10050 has been refined to 2.0A resolution. The electron density has been significantly improved, compared to the 2.5A resolution map, by high resolution data, cryo-cooling and translation, libration, screw (TLS) refinement. The electron density reveals a second carotenoid molecule, the last five C-terminal residues of the alpha-chain and a carboxy modified alpha-Met1 which forms the ligand of the B800 bacteriochlorophyll. TLS refinement has enabled the characterisation of displacements between molecules in the complex. B850 bacteriochlorophyll molecules are arranged in a ring of 18 pigments composed of nine approximate dimers. These pigments are strongly coupled and at their equilibrium positions the excited state dipole interaction energies, within and between dimers, are approximately 370cm(-1) and 280cm(-1), respectively. This difference in coupling energy is similar in magnitude to changes in interaction energies arising from the pigment displacements described by TLS tensors. The displacements appear to be non-random in nature and appear to be designed to optimise the modulation of pigment energy interactions. This is the first time that LH2 pigment displacements have been quantified experimentally. The calculated energy changes indicate that there may be significant contributions to inter-pigment energy interactions from molecular displacements and these may be of importance to photosynthetic energy transfer.

Bacterial Proteins↗

PubMed2000

Helminth C-type lectins and host-parasite interactions.

C-type lectins (C-TLs) are a family of carbohydrate-binding proteins intimately involved in diverse processes including vertebrate immune cell signalling and trafficking, activation of innate immunity in both vertebrates and invertebrates, and venom-induced haemostasis. Helminth C-TLs sharing sequence and structural similarity with mammalian immune cell lectins have recently been identified from nematode parasites, suggesting clear roles for these proteins at the host-parasite interface, notably in immune evasion. Here, Alex Loukas and Rick Maizels review the status of helminth lectin research and suggest ways in which parasitic worms might utilize C-TLs during their life history.

Amino Acid Sequence↗

PubMed1998

Fibroblast-like cells from tendons differ from skin fibroblasts in their ability to form three-dimensional structures in vitro.

Tendon samples cultured in vitro produced cells (TC) with fibroblast-like morphology and confluence occurred within 5 weeks. Histological staining demonstrated proteoglycan and collagen secretion by TC. Immunohistochemical staining revealed type I collagen but no type III. Assay of total collagen demonstrated a rapid increase in synthesis with time in culture. Cultures allowed to become 'superconfluent' spontaneously formed three-dimensional structures after about 4 weeks, which became macroscopic, tendon-like structures (TLS). Cells within TLS seemed under cell-generated tension. Haematoxylin and eosin staining of sections of tendon, of TLS and of TC cultures demonstrated similarities in morphology. These studies were performed using human and rabbit cells and findings were similar for the two species, but with some differences in cell metabolism. Skin fibroblasts were also cultured as a comparison.

Animals↗

PubMed1994

High dialysate flow rate continuous arteriovenous hemodialysis: a new approach for the treatment of acute renal failure and tumor lysis syndrome.

A continuous dialysis technique such as continuous arteriovenous hemodialysis (CAVHD) could be an interesting alternative to frequent intermittent hemodialysis to treat acute renal failure (ARF) secondary to tumor lysis syndrome (TLS). However, because of massive release of intracellular solutes in TLS, CAVHD clearances need to be increased to treat this syndrome. Continuous arteriovenous hemodialysis using a high dialysate flow rate at 4 L/hr was assessed in TLS and ARF associated with severe hyperphosphatemia. A 0.6-m2 hollow-fiber polyacrylonitrile dialyzer (Multiflow 60; Hospal, St-Léonard, Québec, Canada) was used. Blood urea nitrogen and serum creatinine levels decreased, respectively, from 102.5 to 27.2 mg/dL and from 3.1 to 1.8 mg/dL during the 36 hours of treatment. Serum urate concentration was normal at the beginning of treatment (4.5 mg/dL) and decreased to 2.1 mg/dL by the end of CAVHD. Serum phosphorus decreased from 16.7 to 4.4 mg/dL after the 36 hours of treatment. The calcium x phosphorus product decreased from 111.1 to 42.1 by 28 hours and remained under 50 thereafter. Serum potassium was easily controlled with the addition of 2.5 mEq/L of KCl in dialysate and replacement solutions. No rebound increases in phosphorus or potassium were noted after cessation of therapy. Continuous arteriovenous hemodialysis clearances of urea, creatinine, phosphorus, and urate were measured at 2-hour intervals for the first 24 hours and at 4-hour intervals for the remaining 12 hours. They were 53.0 +/- 2.3 mL/min, 43.7 +/- 2.2 mL/min, 40.4 +/- 1.9 mL/min, and 39.3 +/- 1.9 mL/min (n = 15), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Kidney Injury↗