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PubMed2026

Effect of Tertiary Lymphoid Structures on Immune Cell Infiltration in the Tumor Microenvironment and Prognosis in Lung Adenocarcinoma.

Tertiary lymphoid structures (TLSs) modulate immune responses in various solid tumors, but their comprehensive role in lung adenocarcinoma (LUAD) remains unclear. In this study, we analyzed RNA-seq data from 539 LUAD patients in The Cancer Genome Atlas (TCGA) and microarray data from 223 samples from the Gene Expression Omnibus (GEO, GSE13213, and GSE37745). TLS signatures were evaluated via unsupervised consensus clustering based on 12 chemokine transcriptome signatures. The relationships between TLS and clinical characteristics, tumor microenvironment (TME) cell infiltration, and prognosis were assessed using ESTIMATE and CIBERSORT. A prognostic model was established using LASSO regression and validated with external datasets. Additionally, H&E and IHC analyses were performed to explore associations between intratumoral TLS density, immune-related molecular expression, and patient prognosis in LUAD. Consensus clustering of the TCGA cohort revealed two distinct LUAD patient clusters according to TLS abundance. Cluster 1 exhibited greater immune cell infiltration, more favorable prognosis, and increased expression of immune checkpoint molecules. We developed a prognostic model comprising eight survival-associated genes that act as independent prognostic factors for patient survival. H&E/IHC analyses revealed that TLS density-regardless of pathological stage-was associated with better prognosis; higher intratumoral TLS density/proportion was also related to more favorable outcomes. IHC confirmed that survival-associated genes (CD5, HLA-DMB, and P2RY13) are independent prognostic indicators in LUAD. Our study demonstrated the close relationship between TLS signatures and an active immune microenvironment, highlighting their potential as independent prognostic indicators in LUAD.

Humans↗

PubMed1999

SOS mutagenesis results from up-regulation of translesion synthesis.

Irradiation of DNA with ultraviolet light generates a variety of photolesions. Among them, are cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts blocking lesions that interfere with DNA replication if left unrepaired. In addition to efficient pre-replicative excision repair mechanisms, cells have evolved damage tolerance pathways enabling them to replicate lesion-containing DNA molecules either by directly replicating through the damaged base (translesion synthesis, TLS) or by employing the locally undamaged complementary strand thus avoiding the lesion (damage avoidance pathways, DA). Using double-stranded vectors with a single T(6-4)T UV lesion and a strand segregation analysis (SSA), we have measured the relative utilization of the two tolerance pathways (TLS and DA) in Escherichia coli. During the SOS response the error-prone TLS pathway is strongly stimulated ( approximately 20-fold) at the expense of the error-free DA pathways. Thus, up-regulation of TLS may turn out to be a general property of the SOS response; a similar conclusion was previously reached with the frameshift-inducing N-2-acetylaminofluorene adduct. Therefore, as far as its contribution to damaged DNA replication is concerned, the SOS response appears to be an induced mutator state rather than a survival strategy. Depending on the base inserted opposite the lesion, TLS can be error-free or mutagenic. In a wild-type strain, both forms of TLS are increased to a similar extent during the SOS response. In contrast, in a DeltaumuDC strain induction of TLS is totally abolished, demonstrating that the UmuDC proteins usually thought to be specifically involved in mutagenesis facilitate the recovery of both error-free and mutagenic replication intermediates in vivo.

DNA↗

PubMed2005

DNA polymerases eta and kappa are responsible for error-free translesion DNA synthesis activity over a cis-syn thymine dimer in Xenopus laevis oocyte extracts.

In translesion synthesis (TLS), specialized DNA polymerases (pols) facilitate progression of replication forks stalled by DNA damage. Although multiple TLS pols have been identified in eukaryotes, little is known about endogenous TLS pols and their relative contributions to TLS in vivo because of their low cellular abundance. Taking advantage of Xenopus laevis oocyte cells, with their extraordinary size and abundant enzymes involved in DNA metabolism, we have identified and characterized endogenous TLS pols for DNA damage induced by ultraviolet (UV) irradiation. We designed a TLS assay which monitors primer elongation on a synthetic oligomer template over a single UV-induced lesion, either a cys-syn cyclobutane pyrimidine dimer (CPD) or a pyrimidine (6-4) pyrimidone photoproduct. Four distinct TLS activities (TLS1-TLS4) were identified in X. laevis oocyte extracts, using three template/primer (T/P) DNA substrates having various sites at which primer extension is initiated relative to the lesion. TLS1 and TLS2 activities appear to be sequence-dependent. TLS3 and TLS4 extended the primers over the CPD in an error-free manner irrespective of sequence context. Base insertion opposite the CPD of the T/P substrate in which the 3'-end of the primer is placed one base upstream of the lesion was observed only with TLS3. TLS3 and TLS4 showed primer extension with similar efficiencies on the T/P substrate whose 3'-primer terminal dinucleotide (AA) was complementary to the CPD lesion. Investigations with antibodies and recombinant pols revealed that TLS3 and TLS4 were most likely attributable to pol eta and pol kappa, respectively. These results indicate that error-free insertion in CPD bypass is due mainly to pol eta (TLS3) in the extracts, and suggest that pol kappa (TLS4) may assist pol eta (TLS3) in error-free extension during CPD bypass.

Animals↗

PubMed2001

Tumor lysis syndrome.

Tumor lysis syndrome (TLS) is a constellation of metabolic disturbances observed in tumors with high cell turnover. It is associated with significant morbidity and mortality. TLS is characterized by the increased release of intracellular contents (uric acid, potassium, phosphorus) into the extracellular compartment, which can overwhelm the body's capacity for clearance. TLS is usually caused by response to chemotherapy; however, it may also occur spontaneously. Because uric acid, potassium, and phosphorus are excreted primarily by the kidneys, TLS can lead to hyperuricemia, hyperkalemia, and hyperphosphatemia with accompanying renal compromise. The pathophysiology of TLS-associated acute renal failure is probably multifactorial. Potential etiologies include intravascular volume depletion, urinary precipitation of nucleic acid metabolites and calcium phosphate, and malignancy-associated nephrotoxins. Despite prophylactic therapy with allopurinol and volume repletion, patients may still develop TLS with acute renal failure. While reducing the risk of uric acid precipitation, allopurinol and alkalinization increase the risk of xanthine and calcium phosphate crystals, respectively. Aggressive hydration might lead to volume overload, specifically in older patients. Novel approaches in the management of TLS include the use of urate oxidase, which can provide effective treatment with an acceptable safety profile.

Allopurinol↗

PubMed2002

Translesion synthesis by human DNA polymerase eta across thymine glycol lesions.

The XP-V (xeroderma pigmentosum variant) gene product, human DNA polymerase eta (pol eta), catalyzes efficient and accurate translesion synthesis (TLS) past cis-syn thymine-thymine dimers (TT dimer). In addition, recent reports suggest that pol eta is involved in TLS past various other types of lesion, including an oxidative DNA damage, 8-hydroxyguanine. Here, we compare the abilities of pol alpha and pol eta to replicate across thymine glycol (Tg) using purified synthetic oligomers containing a 5R- or 5S-Tg. DNA synthesis by pol alpha was inhibited at both steps of insertion of a nucleotide opposite the lesion and extension from the resulting product, indicating that pol alpha can weakly contribute to TLS past Tg lesions. In contrast, pol eta catalyzed insertion opposite the lesion as efficient as that opposite undamaged T, while extension was inhibited especially on the 5S-Tg template. Thus, pol eta catalyzed relatively efficient TLS past 5R-Tg than 5S-Tg. To compare the TLS abilities of pol eta for these lesions, we determined the kinetic parameters of pol eta for catalyzing TLS past a TT dimer, an N-2-acetylaminofluorene-modified guanine, and an abasic site analogue. The possible mechanisms of pol eta-catalyzed TLS are discussed on the basis of these results.

Base Sequence↗

PubMed1999

Horseshoe crab acetyl group-recognizing lectins involved in innate immunity are structurally related to fibrinogen.

We have characterized and cloned newly isolated lectins from hemolymph plasma of the horseshoe crab Tachypleus tridentatus, which we named tachylectins 5A and 5B (TLs-5). TLs-5 agglutinated all types of human erythrocytes and Gram-positive and Gram-negative bacteria. TLs-5 specifically recognize acetyl group-containing substances including noncarbohydrates; the acetyl group is required and is sufficient for recognition. TLs-5 enhanced the antimicrobial activity of a horseshoe crab-derived big defensin. cDNA sequences of TLs-5 indicated that they consist of a short N-terminal Cys-containing segment and a C-terminal fibrinogen-like domain with the highest sequence identity (51%) to that of mammalian ficolins. TLs-5, however, lack the collagenous domain found in a kind of "bouquet arrangement" of ficolins and collectins. Electron microscopy revealed that TLs-5 form two- to four-bladed propeller structures. The horseshoe crab is equipped with a unique functional homologue of vertebrate fibrinogen, coagulogen, as the target protein of the clotting cascade. Our observations clearly show that the horseshoe crab has fibrinogen-related molecules in hemolymph plasma and that they function as nonself-recognizing lectins. An ancestor of fibrinogen may have functioned as a nonself-recognizing protein.

ABO Blood-Group System↗

PubMed2006

A predictive model for the detection of tumor lysis syndrome during AML induction therapy.

Tumor lysis syndrome (TLS) is defined by metabolic derangements occurring in the setting of rapid tumor destruction. In acute myelogenous leukemia (AML), TLS frequency, risk stratification, monitoring, and management strategies are based largely on case series and data from other malignancies. A single-center, retrospective cohort study was conducted to estimate TLS incidence and identify TLS predictive factors in a patient population undergoing myeloid leukemia induction chemotherapy. This study included 194 patients, aged 18-86 years, with AML or advanced myelodysplastic syndrome undergoing primary myeloid leukemia induction chemotherapy. Nineteen patients (9.8%) developed TLS. In univariate analysis, elevated pre-chemotherapy values for uric acid (P < 0.0001), creatinine (P = 0.0025), lactate dehydrogenase (LDH) (P = 0.0001), white blood cell (P = 0.0058), gender (P = 0.0064) and chronic myelomonocytic leukemia history (P = 0.0292) were significant predictors. In multivariate analysis, LDH (P = 0.0042), uric acid (P < 0.0001) and gender (P = 0.0073) remained significant TLS predictors. A predictive model was then designed using a scoring system based on these factors. This analysis may lay the groundwork for the development of the first evidence-based guidelines for TLS monitoring and management in this patient population.

Adolescent↗

PubMed1996

The effects of clathrin inactivation on localization of Kex2 protease are independent of the TGN localization signal in the cytosolic tail of Kex2p.

Localization of Kex2 protease (Kex2p) to the yeast trans-Golgi network (TGN) requires a TGN localization signal (TLS) in the Kex2p C-terminal cytosolic tail. Mutation of the TLS accelerates transport of Kex2p to the vacuole by an intracellular (SEC1-independent) pathway. In contrast, inactivation of the clathrin heavy-chain gene CHC1 results in transport of Kex2p and other Golgi membrane proteins to the cell surface. Here, the relationship of the two localization defects was assessed by examining the effects of a temperature-sensitive CHC1 allele on trafficking of wild-type (WT) and TLS mutant forms of Kex2p. Inactivation of clathrin by shifting chc1-ts cells to 37 degrees C caused WT and TLS mutant forms of Kex2p to behave identically. All forms of Kex2p appeared at the plasma membrane within 30-60 min of the temperature shift. TLS mutant forms of Kex2p were stabilized, their half-lives increasing to that of wild-type Kex2p. After inactivation of clathrin heavy chain, vacuolar protease-dependent degradation of all forms of Kex2p was blocked by a sec1 mutation, which is required for secretory vesicle fusion to the plasma membrane, indicating that transport to the cell surface was required for degradation by vacuolar proteolysis. Finally, after clathrin inactivation, all forms of Kex2p were degraded in part by a vacuolar protease-independent pathway. After inactivation of both chc1-ts and sec1-ts, Kex2 was degraded exclusively by this pathway. We conclude that the effects of clathrin inactivation on Kex2p localization are independent of the Kex2p C-terminal cytosolic tail. Although these results neither prove nor rule out a direct interaction between the Kex2 TLS and a clathrin-dependent structure, they do imply that clathrin is required for the intracellular transport of Kex2p TLS mutants to the vacuole.

Biological Transport↗

PubMed2003

Prospective validation of computed tomographic screening of the thoracolumbar spine in trauma.

OBJECTIVE: Concern for thoracolumbar spine (TLS) injuries after major trauma mandates immobilization pending radiographic evaluation. Current protocols use standard posteroanterior and lateral radiographs of the thoracolumbar spine (XR/TLS), but many patients also undergo abdominal or thoracic computed tomographic (CT) scanning. We sought to evaluate whether helical truncal CT scanning performed to evaluate visceral trauma images the spine as well as dedicated XR/TLS. METHODS: We prospectively studied 222 consecutive patients sustaining high-risk trauma requiring TLS screening because of clinical findings or altered mentation. The chest, abdomen, and pelvis were imaged with one intravenous contrast infusion. All patients had CT scan of the chest, abdomen, and pelvis (CT/CAP) and XR/TLS. Initial radiologic diagnoses were compared with the discharge diagnosis of acute fractures confirmed by thin-cut CT scan and/or clinical examination of the patient when alert. RESULTS: Of 222 patients studied, 215 were fully evaluated. Thirty-six (17%) had acute TLS fractures. The accuracy of CT/CAP for TLS fractures was 99% (95% confidence interval [CI], 96-100%). The accuracy of XR/TLS was 87% (95% CI, 82-92%). Sensitivity, specificity, and positive and negative predictive values were better for CT/CAP than for XR/TLS. CT/CAP found acute fractures XR/TLS missed, and correctly classified old fractures XR/TLS read as "possibly" acute. The total XR/TLS misclassification rate was 12.6% (95% CI, 8.4-19%); for CT/CAP it was 1.4% (95% CI, 0.3-3.3%). No fractures were missed by CT/CAP. No unstable fracture was missed by either technique. CONCLUSION: CT/CAP diagnoses TLS fractures more accurately than XR/TLS. Neither misses unstable fractures, but CT scanning finds small fractures that benefit by treatment and identifies chronic disease better. CT screening is far faster and shortens time to removal of spine precautions. CT scan-based diagnosis does not result in greater radiation exposure and improves resource use. Screening the TLS on truncal helical CT scanning performed for the evaluation of visceral injuries is more accurate than TLS imaging by standard radiography. CT/CAP should replace plain radiographs in high-risk trauma patients who require screening.

Adult↗

PubMed1995

Genetic regulation of development of thymic lymphomas induced by N-propyl-N-nitrosourea in the rat.

To clarify the linkage between Hbb and Tls-1 (thymic lymphoma susceptible-1) loci and to investigate other loci concerned in thymic lymphomagenesis, the BUF/Mna rat, which is highly sensitive to the lymphomagenic activity of N-propyl-N-nitrosourea (PNU), the WKY/NCrj rat, reported to be resistant, and their cross offspring were subjected to genetic analysis. F1 hybrid and backcross generations were raised from the 2 strains, and 6 genetic markers including Hbb were analyzed in individuals of the backcross generation. However, no linkage between Hbb and Tls-1 loci could be demonstrated since WKY rats also developed a high incidence of thymic lymphomas in response to PNU. Nevertheless, thymic lymphomas developed more rapidly and reached a larger size in the BUF rats. F1 rats expressed a rather rapid and large tumor growth phenotype, while the [(WKY X BUF) X WKY] backcross generation consisted of rats with either rapidly growing or slowly growing tumors. It was thus concluded that rapid development of thymic lymphomas is determined by a gene, provisionally designated Tls-3. Analysis of the relationship between 6 genetic markers and development of thymic lymphoma in the backcross generation demonstrated that the Tls-3 locus is loosely linked to the Gc locus, suggesting a possible location on rat chromosome 14. Tls-3 may not be identical with Tls-1 and other genes known to be relevant to thymic tumors, but its relationship with Tls-2 remains obscure.

Animals↗

PubMed1989

Temporal lobe syncope: clinical variants.

Temporal lobe syncope (TLS) is a term coined by Landolt. Characteristically, the patient has psychomotor and drop attacks, and the interictal electroencephalogram (EEG) shows temporal lobe epileptic abnormalities. TLS is synonymous with type III complex partial seizures (CPSs) in the Delgado Escueta classification. Several variants of TLS can be recognized including atonic akinetic, simple akinetic, atonic, atonic-tonic complex (automatisms), sexual seizures, stress-induced convulsions, and gelastic atonic seizures. TLS must be distinguished from drop attacks of vertebrobasilar insufficiency and associated EEG abnormalities, and from hereditary tachyarrhythmias mimicking stress-induced convulsions. Epileptic falls and drop attacks are discarded by ictal EEG recordings. Recognition of TLS variants is important in the prospective evaluation of the surgical treatment of epilepsy given the past conflicting reports on the differential outcome of surgically treated CPSs. TLS is an attractive clinical term, easy to remember, and with pathophysiologic relevance to the clinician confronting the patient with a history of syncope and whose EEG discloses temporal lobe paroxysmal activity. The detailed ictal electrophysiology of TLS is unknown.

Adult↗

PubMed2003

Role of i.v. allopurinol and rasburicase in tumor lysis syndrome.

The role of i.v. allopurinol and rasburicase in tumor lysis syndrome (TLS) is described. The current standard management for TLS consists of oral allopurinol in conjunction with i.v. hydration with or without alkalinization. Despite this standard prophylactic regimen, some high-risk patients may still develop urate nephropathy from TLS. Recently, i.v. allopurinol and rasburicase became available for the management of TLS. Available data on i.v. allopurinol indicate that the administration schedule and the adverse-effect profile will be similar to the oral formulation. The primary advantage of i.v. allopurinol is the flexibility of administration for patients who cannot take anything by mouth, since there are no data indicating the superiority of the i.v. to the oral product. Rasburicase is the first agent that will oxidize uric acid to allantoin, a metabolite with 5-10-fold greater solubility than uric acid, and reduces serum uric acid (SUA) levels within four hours of administration. Rasburicase is considerably more expensive than standard management strategies and should be reserved for patients with either renal dysfunction, significant elevations in SUA values, or large tumor burdens. Preliminary evidence indicates that rasburicase offers cost savings in the treatment of TLS and is cost-effective as a strategy for preventing TLS for many cancer patients. Both i.v. allopurinol and rasburicase offer additional flexibility in the management of TLS and may allow for further avoidance of the consequences of inadequate management of this syndrome.

Administration, Oral↗

PubMed1997

Effect of exogenous growth hormone on in vitro proliferation of thymic lymphocytes from the hypophysectomized rats.

Thymus weight and total thymic lymphocyte (TL) number decreased markedly after hypophysectomy (HX). Serum level of insulin-like growth factor-I (IGF-I), one of mediators brought about by growth hormone (GH), reduced considerably after the operation. Exogenous GH stimulation enhanced significantly DNA synthetic activity of TLs from HX group only at lower cell concentrations, less than 5 x 10(5) cell/ml. However, there was not a significant difference in the percentage increase of the TL-DNA synthetic activity with exogenous GH between HX and sham-operated (SO) groups. Since GH-responding TLs appeared to be mature cells and to exist in thymus medulla, the results suggest that rate of the responding cells in the thymus medulla of HX animals is similar to that of SO ones. Furthermore, there was not a significant difference in the percentages of rosette-forming cells and non-rosette-forming cells, reflecting nearly the maturation steps of TLs, between groups. It is difficult to explain such an aspect in spite of severe reduction of TLs after hypophysectomy, since the proliferative-responding rate of TLS to exogenous GH stimulation and the constitutive ratio of each maturation step of TLs in the HX group were almost same as those of SO group. The contradictory data found in thymus after hypophysectomy are discussed from the point of view of the compensatory effects of growth hormone-releasing hormone on TLs from the operated animals.

Animals↗

PubMed2001

Decorin inhibits endothelial migration and tube-like structure formation: role of thrombospondin-1.

Interactions between endothelial cell receptors and the extracellular matrix (ECM) play a critical, yet poorly understood role in angiogenesis. Based on the anti-adhesive role of decorin, we hypothesized that decorin binding to ECM molecules such as thrombospondin-1 (TSP-1) plays a regulatory role in endothelial tube-like structure (TLS) formation. To test this hypothesis, endothelial cells were plated on TSP-1, decorin, or mixed substrates of TSP-1 plus decorin. TLS formation was induced by applying type I collagen on the confluent endothelial monolayer. Cartilage decorin inhibited the formation of TLSs in a concentration-dependent manner. On substrates of high decorin concentrations (2.5 and 5.0 microg/cm(2)) the reduction in TLSs was due either to a reduction in the number of adhering cells or to decreased cell migration. At low decorin concentrations (0.05 and 0.25 microg/cm(2)) the reduction in TLSs was independent of the number of attached cells. Time-lapse video microscopy revealed that decorin substrates facilitated homotypic aggregation and isolated cord formation at the expense of endothelial migration and TLS formation. Consistent with the reduced migration, endothelial cells formed fewer vinculin-positive focal adhesions and actin-stress fibers on decorin substrates. Endothelial migration and TLS formation were also significantly inhibited by skin decorin and the protein core of cartilage decorin. The inhibition of TLS formation by the protein core of cartilage decorin was potentiated by TSP-1. These findings suggest that decorin alone or in combination with TSP-1 interferes with the activation of endothelial cell receptors by ECM molecules, thus blocking intracellular signals that induce cytoskeletal reorganization, migration, and TLS formation.

Cartilage↗

PubMed2000

Timed loaded standing: a measure of combined trunk and arm endurance suitable for people with vertebral osteoporosis.

Chronic back tiredness or fatigue is a common complaint of people who have a history of osteoporotic vertebral fracture. Trunk muscle endurance has not been studied in people with vertebral osteoporosis, partly due to the lack of assessment tools. We developed a measure of combined trunk and arm endurance suitable for people with vertebral osteoporosis, timed loaded standing (TLS). TLS measures the time a person can stand while holding a two-pound dumbbell in each hand with the arms at 90 degrees of shoulder flexion and the elbows extended. Intraclass correlation coefficients (ICCs) for same day inter-trial and six to ten day test-retest reliability were 0.89 (lower bound 95% confidence interval [LB 95% CI] 0.79) and 0.84 (LB 95% CI 0.68), respectively, in a sample of 21 older women with no known osteoporosis. In 127 women with vertebral fractures, the ICC for same day inter-trial reliability was 0.81 (LB 95% CI 0.75). In a sub-sample of 30 of these women with vertebral fractures, the six to ten day test-retest reliability was 0.85 (LB 95% CI 0.75). Moderately strong and statistically significant (p < or = 0.05) correlations were found between TLS and sixteen of eighteen measures of physical impairment and function. Functional reach distance, gait velocity, MOS-36 Physical Function Subscale, shoulder flexion strength, and six minute walk distance were most strongly associated with TLS time. Women with vertebral fractures who endorsed having back tiredness when standing and working with the arms in front of the body, sitting to rest because of back tiredness or pain, and planning rest periods because of back tiredness or pain had significantly lower TLS times. TLS is a simple, safe physical performance measure of combined trunk and arm endurance that demonstrates acceptable reliability (inter-trial and test- retest) and concurrent validity.

Aged↗

PubMed1982

Influence of strain difference on the karyotypic changes in N-nitroso-N-butylurea--induced mouse lymphomas.

Karyotypes were analyzed in 44 cases of mouse lymphomas induced by continuous oral administration of N-nitroso-N-butylurea (NBU): 14 cases of C57BL/6JKok, 6 from LT/sv, 5 from MT/Hok, 2 from (AKR/MsHok x T1Ct)F1, 2 from (AKR/MsHok x CBA/H-T6Hok)F1, and 15 from the first backcross generation of (CBA/Hok x CBA/H-T6Hok)F1 to CBA/Hok. Thirty-six were thymic lymphomas (TLs) and the remaining 8 nonthymic lymphomas (NTLs). No chromosome abnormality was found in 8 NTLs, whereas modal cells were karyotypically changed in 13 TLs. Unlike the situation in NTLs, chromosomally abnormal cells were always present in the remaining 23 TL cases in which modal cells showed no apparent deviation from the constitutional host karyotype. A total of 31 independent karyotypic changes were identified in 26 cytogenetically abnormal major clones from 19 TLs. Although trisomy 15 was the most frequent karyotypic change, being present in 9 TLs, its incidence was substantially low compared with experimentally induced murine TLs so far reported. Furthermore, 6 of the 9 trisomy 15 clones clustered to (CBA x T6)F1 x CBA. Thus, it seems probable that the genetic background of the host animals has a profound influence upon the frequency of trisomy 15 in TLs.

Animals↗

PubMed2003

Altered translesion synthesis in E. coli Pol V mutants selected for increased recombination inhibition.

Replication of damaged DNA, also termed as translesion synthesis (TLS), involves specialized DNA polymerases that bypass DNA lesions. In Escherichia coli, although TLS can involve one or a combination of DNA polymerases depending on the nature of the lesion, it generally requires the Pol V DNA polymerase (formed by two SOS proteins, UmuD' and UmuC) and the RecA protein. In addition to being an essential component of translesion DNA synthesis, Pol V is also an antagonist of RecA-mediated recombination. We have recently isolated umuD' and umuC mutants on the basis of their increased capacity to inhibit homologous recombination. Despite the capacity of these mutants to form a Pol V complex and to interact with the RecA polymer, most of them exhibit a defect in TLS. Here, we further characterize the TLS activity of these Pol V mutants in vivo by measuring the extent of error-free and mutagenic bypass at a single (6-4)TT lesion located in double stranded plasmid DNA. TLS is markedly decreased in most Pol V mutants that we analyzed (8/9) with the exception of one UmuC mutant (F287L) that exhibits wild-type bypass activity. Somewhat unexpectedly, Pol V mutants that are partially deficient in TLS are more severely affected in mutagenic bypass compared to error-free synthesis. The defect in bypass activity of the Pol V mutant polymerases is discussed in light of the location of the respective mutations in the 3D structure of UmuD' and the DinB/UmuC homologous protein Dpo4 of Sulfolobus solfataricus.

DNA↗

PubMed2005

Dispensability of 3' tRNA-like sequence for packaging cowpea chlorotic mottle virus genomic RNAs.

The 3' ends of three genomic RNAs (gRNAs) of cowpea chlorotic mottle virus (CCMV) terminate in a highly conserved tRNA-like structure (3'TLS). To examine the intrinsic role played the 3'TLS in packaging, the competence of each gRNA lacking the 3' TLS (DeltaTLS-gRNA) to interact with dissociated coat protein (CP) subunits and form virions was assayed in vitro. In contrast to the well established requirement for the participation of either viral 3'TLS or host-tRNAs in the assembly of RNA-containing virions in brome mosaic virus (BMV; Choi, Y, G., Dreher, T. W., Rao, A. L. N. 2002. tRNA elements mediate the assembly of an icosahedral RNA virus. Proc. Natl. Acad. Sci. 99, 655-660), CCMV CP does not require the presence of viral TLS in cis or in trans. Similar in vitro assembly assays showed that CCMV CP subunits also packaged BMV RNAs lacking 3' TLS as well as two other non-bromoviral RNAs although with lesser efficiency. To characterize sequences of CCMV RNA3 (C3) required for packaging, a series deletions was engineered into C3 and their effect on virus assembly was examined. It was observed that, unlike BMV RNA3 whose packaging requires a bipartite signal (Choi, Y. G., Rao, A. L. N. 2003. Packaging of brome mosaic virus RNA3 is mediated through a bipartite signal. J. Virol. 77, 9750-9757), packaging of C3 is independent of either movement protein (MP) ORF or CP ORF or 3' non-coding regions. Based on the differential prerequisites identified in this study for the assembly of BMV and CCMV, we hypothesize that the adaptive condition for movement in monocotyledonous host has made packaging a necessary co-requirement for BMV.

3' Untranslated Regions↗