Pancreatic cancer biomarkers: A pathway to advance in personalized treatment selection.

Pancreatic cancer is one of the tumors with the worst prognosis, and unlike other cancers, few advances have been made in recent years. The only curative option is surgery, but only 15-20% of patients are candidates, with a high risk of relapse. In advanced pancreatic cancer there are few first-line treatment options and no validated biomarkers for better treatment selection. The development of targeted therapies in pancreatic cancer is increasingly feasible due to tumor-agnostic treatments, such as PARP inhibitors in patients with BRCA1, BRCA2 or PALB2 alterations or immunotherapies in patients with high microsatellite instability/tumor mutational burden. In addition, other therapeutic molecules have been developed for patients with KRAS G12C mutation or fusions in NTRK or NRG1. Consequently, there has been a growing interest in biomarkers that may help guide targeted therapy in pancreatic cancer. Therefore, this review aims to offer an updated perspective on biomarkers with therapeutic potential in pancreatic cancer.

Clinical significance of glycogen synthase kinase 3 (GSK-3) expression and tumor budding grade in colorectal cancer: Implications for targeted therapy.

INTRODUCTION: Glycogen synthase kinase 3 (GSK-3) has been proposed as a novel cancer target due to its regulating role in both tumor and immune cells. However, the connection between GSK-3 and immunoevasive contexture, including tumor budding (TB) has not been previously examined. METHODS: we investigated the expression levels of total GSK-3 as well as its isoforms (GSK-3ß and GSK-3α) and examined their potential correlation with TB grade and the programmed cell death-ligand 1 (PD-L1) in colorectal cancer (CRC) tumor samples. Additionally, we compared the efficacy of GSK-3-inhibition with PD-1/PD-L1 blockade in humanized patient-derived (PDXs) xenografts models of high-grade TB CRC. RESULTS: we show that high-grade (BD3) TB CRC is associated with elevated expression levels of total GSK-3, specifically the GSK-3ß isoform, along with increased expression of PD-L1 in tumor cells. Moreover, we define an improved risk stratification of CRC patients based on the presence of GSK-3+/PD-L1+/BD3 tumors, which are associated with a worse prognosis. Significantly, in contrast to the PD-L1/PD-1 blockade approach, the inhibition GSK-3 demonstrated a remarkable enhancement in the antitumor response. This was achieved through the reduction of tumor buds via necrosis and apoptosis pathways, along with a notable increase of activated tumor-infiltrating CD8+ T cells, NK cells, and CD4- CD8- T cells. CONCLUSIONS: our study provides compelling evidence for the clinical significance of GSK-3 expression and TB grade in risk stratification of CRC patients. Moreover, our findings strongly support GSK-3 inhibition as an effective therapy specifically targeting high-grade TB in CRC.

Diagnosing and monitoring pancreatic cancer through cell-free DNA methylation: progress and prospects.

Pancreatic cancer is one of the most challenging cancers due to its high mortality rates. Considering the late diagnosis and the limited survival benefit with current treatment options, it becomes imperative to optimize early detection, prognosis and prediction of treatment response. To address these challenges, significant research efforts have been undertaken in recent years to develop liquid-biopsy-based biomarkers for pancreatic cancer. In particular, an increasing number of studies point to cell-free DNA (cfDNA) methylation analysis as a promising non-invasive approach for the discovery and validation of epigenetic biomarkers with diagnostic or prognostic potential. In this review we provide an update on recent advancements in the field of cfDNA methylation analysis in pancreatic cancer. We discuss the relevance of DNA methylation in the context of pancreatic cancer, recent cfDNA methylation research, its clinical utility, and future directions for integrating cfDNA methylation analysis into routine clinical practice.

Circulating NPTX2 methylation as a non-invasive biomarker for prognosis and monitoring of metastatic pancreatic cancer.

BACKGROUND: Pancreatic cancer is the most lethal cancer with a dismal prognosis mainly due to diagnosis at advanced stage and ineffective treatments. CA19-9 levels and computed tomography (CT) imaging are the main standard criteria for evaluating disease progression and treatment response. In this study we explored liquid biopsy-based epigenetic biomarkers for prognosis and monitoring disease in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC). METHODS: Plasma samples were collected from 44 mPDAC patients at the time of diagnosis, and in 15 of them, additional samples were obtained during follow-up of the disease. After cell-free DNA (cfDNA), isolation circulating levels of methylated NPTX2, SPARC, BMP3, SFRP1 and TFPI2 genes were measured using digital droplet PCR (ddPCR). BEAMing technique was performed for quantitation of RAS mutations in cfDNA, and CA19-9 was measured using standard techniques. RESULTS: NPTX2 was the most highly and frequently methylated gene in cfDNA samples from mPDAC patients. Higher circulating NPTX2 methylation levels at diagnosis were associated with poor prognosis and efficiently stratified patients for prediction of overall survival (6.06% cut-off, p = 0.0067). Dynamics of circulating NPTX2 methylation levels correlated with disease progression and response to therapy and predicted better than CA19-9 the evolution of disease in mPDAC patients. Remarkably, in many cases the disease progression detected by CT scan was anticipated by an increase in circulating NPTX2 methylation levels. CONCLUSIONS: Our study supports circulating NPTX2 methylation levels as a promising liquid biopsy-based clinical tool for non-invasive prognosis, monitoring disease evolution and response to treatment in mPDAC patients.

Metabolic shift underlies tumor progression and immune evasion in S-nitrosoglutathione reductase-deficient cancer.

S-nitrosoglutathione reductase (GSNOR) is a denitrosylase enzyme that has been suggested to play a tumor suppressor role, although the mechanisms responsible are still largely unclear. In this study, we show that GSNOR deficiency in tumors is associated with poor prognostic histopathological features and poor survival in patients with colorectal cancer (CRC). GSNOR-low tumors were characterized by an immunosuppressive microenvironment with exclusion of cytotoxic CD8+ T cells. Notably, GSNOR-low tumors exhibited an immune evasive proteomic signature along with an altered energy metabolism characterized by impaired oxidative phosphorylation (OXPHOS) and energetic dependence on glycolytic activity. CRISPR-Cas9-mediated generation of GSNOR gene knockout (KO) CRC cells confirmed in vitro and in vivo that GSNOR-deficiency conferred higher tumorigenic and tumor-initiating capacities. Moreover, GSNOR-KO cells possessed enhanced immune evasive properties and resistance to immunotherapy, as revealed following xenografting them into humanized mouse models. Importantly, GSNOR-KO cells were characterized by a metabolic shift from OXPHOS to glycolysis to produce energy, as indicated by increased lactate secretion, higher sensitivity to 2-deoxyglucose (2DG), and a fragmented mitochondrial network. Real-time metabolic analysis revealed that GSNOR-KO cells operated close to their maximal glycolytic rate, as a compensation for lower OXPHOS levels, explaining their higher sensitivity to 2DG. Remarkably, this higher susceptibility to glycolysis inhibition with 2DG was validated in patient-derived xenografts and organoids from clinical GSNOR-low tumors. In conclusion, our data support the idea that metabolic reprogramming induced by GSNOR deficiency is an important mechanism for tumor progression and immune evasion in CRC and that the metabolic vulnerabilities associated with the deficiency of this denitrosylase can be exploited therapeutically. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Actitudes hacia las políticas sociales: de indicadores individuales a constructos latentes

El objetivo de este estudio fue avanzar hacia la identificación de dimensiones latentes a partir de una serie de ítems comúnmente utilizados en la literatura sobre políticas sociales con relevancia local. Específicamente, se analizó la evidencia de validez estructural y de consistencia interna de un conjunto de 24 ítems sobre actitudes hacia políticas sociales. Participaron 442 personas de 18 a 64 años del Gran Córdoba (Argentina). Se evaluaron modelos de manera exploratoria y confirmatoria (dos factores, tres factores, cuatro factores, segundo orden). La evidencia exploratoria no arrojó resultados satisfactorios. En los análisis confirmatorios el modelo de cuatro factores mostró un ajuste aceptable, con el siguiente agrupamiento de ítems: políticas focalizadas centradas en el beneficiario que tienden a la asistencia (7 ítems), políticas focalizadas centradas en el beneficiario que tienden a la promoción (6 ítems), políticas focalizadas centradas en el contribuyente (4 ítems) y políticas universales (7 ítems). Todos los ítems mostraron cargas factoriales superiores a .40. Los valores de consistencia interna fueron superiores a .70. Los resultados evidencian la posibilidad de considerar dimensiones latentes a las actitudes hacia políticas sociales. Se destaca la necesidad de desarrollar trabajos interdisciplinarios y con anclaje local en relación a la temática.

O objetivo deste estudo foi avançar na identificação de dimensões latentes a partir de uma série de itens comumente utilizados na literatura sobre políticas sociais com relevância local. Especificamente, analisamos as evidências de validade estrutural e consistência interna de um conjunto de 24 itens sobre atitudes em relação às políticas sociais. Participaram 442 pessoas de 18 a 64 anos da Grande Córdoba (Argentina). Os modelos foram avaliados de forma exploratória e confirmatória (dois fatores, três fatores, quatro fatores, segunda ordem). Os resultados da evidência exploratória foram insatisfatórios. Em análises confirmatórias, o modelo de quatro fatores mostrou um ajuste aceitável. Esse modelo implica o agrupamento dos itens nas seguintes dimensões: políticas direcionadas ao beneficiário que tendem a cuidar (7 itens), políticas direcionadas ao beneficiário que tendem a promover (6 itens), políticas direcionadas ao contribuinte (4 itens) e políticas universais (7 itens). Todos os itens apresentaram cargas fatoriais superiores a 0,40. Os valores de consistência interna foram maiores que 0,70. Os resultados mostram a possibilidade de considerar dimensões latentes nas atitudes em relação às políticas sociais. Destacamos a necessidade de desenvolver um trabalho interdisciplinar e ancorado localmente.

The objective of this study was to identify latent dimensions of attitudes towards social policies from a series of items commonly used in locally relevant literature on the subject. Specifically, we analysed evidence of structural validity and internal consistency of a set of 24 items on attitudes towards social policies. A total of 442 people aged 18 to 64 from Gran Cordoba (Argentina) participated. Exploratory and confirmatory (two factors, three factors, four factors, second order) models were assessed. The exploratory evidence did not provide satisfactory results. In the confirmatory analyses, the four-factor model showed an acceptable fit, with the following item clustering: targeted beneficiary-centred policies that tend towards social welfare (7 items), targeted beneficiary-centred policies that tend toward social advancement (6 items), targeted contributor-centred policies (4 items), and universal policies (7 items). All items showed factor loadings greater than .40. The internal consistency values were higher than .70. The results showed the possibility of considering latent dimensions on attitudes towards social policies. We underline the need to develop interdisciplinary and contextualized research in this regard.

Analysis of Global and Local DNA Methylation Patterns in Blood Samples of Patients With Autism Spectrum Disorder.

The goal of this investigation was to determine whether there are alterations in DNA methylation patterns in children with autism spectrum disorder (ASD). Material and Methods: Controlled prospective observational case-control study. Within the ASD group, children were sub-classified based on the presence (AMR subgroup) or absence (ANMR subgroup) of neurodevelopmental regression during the first 2 years of life. We analyzed the global levels of DNA methylation, reflected in LINE-1, and the local DNA methylation pattern in two candidate genes, Neural Cell Adhesion Molecule (NCAM1) and Nerve Growth Factor (NGF) that, according to our previous studies, might be associated to an increased risk for ASD. For this purpose, we utilized blood samples from pediatric patients with ASD (n = 53) and their corresponding controls (n = 45). Results: We observed a slight decrease in methylation levels of LINE-1 in the ASD group, compared to the control group. One of the CpG in LINE-1 (GenBank accession no.X58075, nucleotide position 329) was the main responsible for such reduction, highly significant in the ASD subgroup of children with AMR (p < 0.05). Furthermore, we detected higher NCAM1 methylation levels in ASD children, compared to healthy children (p < 0.001). The data, moreover, showed higher NGF methylation levels in the AMR subgroup, compared to the control group and the ANMR subgroup. These results are consistent with our prior study, in which lower plasma levels of NCAM1 and higher levels of NGF were found in the ANMR subgroup, compared to the subgroup that comprised neurotypically developing children. Conclusions: We have provided new clues about the epigenetic changes that occur in ASD, and suggest two potential epigenetic biomarkers that would facilitate the diagnosis of the disorder. We similarly present with evidence of a clear differentiation in DNA methylation between the ASD subgroups, with or without mental regression.

The Combination of Neutrophil-Lymphocyte Ratio and Platelet-Lymphocyte Ratio with Liquid Biopsy Biomarkers Improves Prognosis Prediction in Metastatic Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a highly inflammatory microenvironment and liquid biopsy has emerged as a promising tool for the noninvasive analysis of this tumor. In this study, plasma was obtained from 58 metastatic PDAC patients, and neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), circulating cell-free DNA (cfDNA) concentration, and circulating RAS mutation were determined. We found that NLR was significantly associated with both overall survival (OS) and progression-free survival. Remarkably, NLR was an independent risk factor for poor OS. Moreover, NLR and PLR positively correlated, and combination of both inflammatory markers significantly improved the prognostic stratification of metastatic PDAC patients. NLR also showed a positive correlation with cfDNA levels and RAS mutant allelic fraction (MAF). Besides, we found that neutrophil activation contributed to cfDNA content in the plasma of metastatic PDAC patients. Finally, a multi-parameter prognosis model was designed by combining NLR, PLR, cfDNA levels, RAS mutation, RAS MAF, and CA19-9, which performs as a promising tool to predict the prognosis of metastatic PDAC patients. In conclusion, our study supports the idea that the use of systemic inflammatory markers along with circulating tumor-specific markers may constitute a valuable tool for the clinical management of metastatic PDAC patients.

DNA Methylation Editing by CRISPR-guided Excision of 5-Methylcytosine.

Tools for actively targeted DNA demethylation are required to increase our knowledge about regulation and specific functions of this important epigenetic modification. DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5-meC), which may promote its replication-dependent dilution and/or active removal through base excision repair (BER). However, it is still unclear whether oxidized derivatives of 5-meC are simply DNA demethylation intermediates or rather epigenetic marks on their own. Unlike animals, plants have evolved enzymes that directly excise 5-meC without previous modification. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5-meC DNA glycosylase to a CRISPR-associated null-nuclease (dCas9) and analyzed its capacity for targeted reactivation of methylation-silenced genes, in comparison to other dCas9-effectors. We found that dCas9-ROS1, but not dCas9-TET1, is able to reactivate methylation-silenced genes and induce partial demethylation in a replication-independent manner. We also found that reactivation induced by dCas9-ROS1, as well as that achieved by two different CRISPR-based chromatin effectors (dCas9-VP160 and dCas9-p300), generally decreases with methylation density. Our results suggest that plant 5-meC DNA glycosylases are a valuable addition to the CRISPR-based toolbox for epigenetic editing.

Validating the Structure of the New Ecological Paradigm Scale among Argentine Citizens through Different Approaches / Validez estructural de la Escala del Nuevo Paradigma Ecológico en ciudadanos argentinos utilizando diferentes abordajes / Validade estrutural da Escada do Novo Paradigma Ecológico em cidadãos argentinos usando diferentes abordagens

Objective. This study assessed the psychometric properties of the New Ecological Paradigm - R scale, in a sample of adult people from Córdoba, Argentina. This scale assesses attitudes, beliefs, values, and worldviews regarding the environment. Previous studies have found differences in the dimensionality, while the consistency has shown to be acceptable. Method. The structure of the instrument was explored using an exploratory factor analysis and also an exploratory structural equation modeling. Results. Both techniques have shown that the bifactor model comprising 11 items yielded the best fit to the data compared to other models. Internal consistency was adequate. While gender differences were not observed, a high educational level was associated with a more ecological view, and age was inversely related to it. Conclusion. Even though good psychometric properties were observed, there is a need for further studies in order to obtain evidence of the predictive value of the scale in the Argentinian context.

Objetivo. Este estudio evaluó las propiedades psicométricas de la escala del Nuevo Paradigma Ecológico - R, en una muestra de personas adultas de Córdoba, Argentina. Esta escala evalúa actitudes, creencias, valores y visiones del mundo con respecto al ambiente. Estudios previos han encontrado diferentes estructuras factoriales subyacentes, en tanto que la evidencia de consistencia interna es aceptable. Método. Se analizó la estructura del instrumento por medio de análisis factorial exploratorio y también con un modelo de ecuaciones estructurales exploratorio. Resultados. Ambas técnicas mostraron que el modelo bifactorial que comprende 11 ítems produjo el mejor ajuste a los datos en relación con otros modelos. La consistencia interna fue adecuada. No se observaron diferencias de género, en tanto que un nivel educativo mayor se asoció con una visión más ecológica y la edad se relacionó de manera inversa. Conclusión. Aunque se observaron adecuadas propiedades psicométricas, se resalta la necesidad de realizar futuros estudios para obtener evidencia del valor predictivo de la NEP-R en el contexto argentino.

Escopo. Em este estudo, foram avaliadas as propriedades psicométricas da escalaa do Novo Paradigma Ecológico - R, em uma amostra de pessoas adultas da cidade de Córdoba, Argentina. Esta escala avalia atitudes, crenças, valores e visões de mundo sobre o médio ambiente. Estudos prévios têm achado diferentes estruturas fatoriais subjacentes, em tanto que a evidencia de consistência interna é aceitável. Metodologia. A estrutura do instrumento foi explorada por médio de uma análise fatorial exploratória e também com um modelo de equações estruturais exploratório. Resultados. Ambas técnicas mostraram que o modelo bi-fatorial composto por 11 itens produziu o melhor ajuste aos dados em relação a outros modelos. A consistência interna foi adequada. Não foram observadas diferencias de gênero, enquanto um nível educativo maior foi associado com uma visão mais ecológica e a idade foi relacionada de jeito inverso. Conclusão. Embora não foram observadas boas propriedades psicométricas, remarcamos a necessidade de futuros estudos para obter evidencia de valor preditiva da NEP-R em nosso contexto.

DNA methylation reprogramming of human cancer cells by expression of a plant 5-methylcytosine DNA glycosylase.

Patterns of DNA methylation, an important epigenetic modification involved in gene silencing and development, are disrupted in cancer cells. Understanding the functional significance of aberrant methylation in tumors remains challenging, due in part to the lack of suitable tools to actively modify methylation patterns. DNA demethylation caused by mammalian DNA methyltransferase inhibitors is transient and replication-dependent, whereas that induced by TET enzymes involves oxidized 5mC derivatives that perform poorly understood regulatory functions. Unlike animals, plants possess enzymes that directly excise unoxidized 5mC from DNA, allowing restoration of unmethylated C through base excision repair. Here, we show that expression of Arabidopsis 5mC DNA glycosylase DEMETER (DME) in colon cancer cells demethylates and reactivates hypermethylated silenced loci. Interestingly, DME expression causes genome-wide changes that include both DNA methylation losses and gains, and partially restores the methylation pattern observed in normal tissue. Furthermore, such methylome reprogramming is accompanied by altered cell cycle responses and increased sensibility to anti-tumor drugs, decreased ability to form colonospheres, and tumor growth impairment in vivo. Our study shows that it is possible to reprogram a human cancer DNA methylome by expression of a plant DNA demethylase.

A specific N-terminal extension of the 8 kDa domain is required for DNA end-bridging by human Polµ and Polλ.

Human DNA polymerases mu (Polµ) and lambda (Polλ) are X family members involved in the repair of double-strand breaks in DNA during non-homologous end joining. Crucial abilities of these enzymes include bridging of the two 3' single-stranded overhangs and trans-polymerization using one 3' end as primer and the other as template, to minimize sequence loss. In this context, we have studied the importance of a previously uncharacterised sequence ('brooch'), located at the N-terminal boundary of the Polß-like polymerase core, and formed by Tyr(141), Ala(142), Cys(143), Gln(144) and Arg(145) in Polµ, and by Trp(239), Val(240), Cys(241), Ala(242) and Gln(243) in Polλ. The brooch is potentially implicated in the maintenance of a closed conformation throughout the catalytic cycle, and our studies indicate that it could be a target of Cdk phosphorylation in Polµ. The brooch is irrelevant for 1 nt gap filling, but of specific importance during end joining: single mutations in the conserved residues reduced the formation of two ended synapses and strongly diminished the ability of Polµ and polymerase lambda to perform non-homologous end joining reactions in vitro.

Propiedades Psicométricas de la escala de felicidad subjetiva en jóvenes y adultos de la ciudad de Córdoba - Argentina / Psychometric Properties of the subjective happiness scale among youths and adults from the city of Cordoba - Argentina

La Escala de Felicidad Subjetiva ha sido validada y aplicada en distintos países del mundo. Esta situación es contraria a lo que sucede en el contexto Latinoamericano. Ante ello, se procedió a la traducción de la escala original propuesta por Lyubomirsky y Lepper (1999) y se efectuaron dos estudios con el objetivo de evaluar las propiedades psicométricas del instrumento en muestras de jóvenes universitarios y adultos de 18 a 65 años de la ciudad de Córdoba (Argentina). En el estudio A se investigó la validez de constructo a través de análisis factorial exploratorio y confirmatorio. Además, se evaluó la correlación ítem total y la consistencia interna a través del alfa de Cronbach. En el estudio B se buscó obtener evidencia adicional sobre la estructura factorial y sobre la validez convergente/ discriminante en relación con la Escala de Afectividad Positiva y Negativa, además de examinarse la consistencia interna de la escala. En términos generales, los resultados evidenciaron una estructura unifactorial que explica el 57.38% de la varianza total, estructura que se replicó en análisis confirmatorios con ambas muestras. La consistencia interna fue aceptable en los dos estudios (coeficiente de alfa de Cronbach superior a .68). Asimismo, una mayor puntuación de felicidad subjetiva se relacionó significativamente y en sentido positivo con la afectividad positiva, y en sentido negativo con la afectividad negativa. Estos resultados indican que la escala posee adecuadas propiedades psicométricas, convirtiéndose así en una herramienta útil y confiable para continuar con el estudio de la felicidad.

The Subjective Happiness Scale has been validated and used around the world. However, this situation is not the same in the Latin-American context. For this reason, this study undertook to translate the original Scale developed by Lyubomirsky y Lepper (1999) and carried out two studies in order to examine the psychometric properties of the scale in samples of students and adults aged 18 to 65 from Cordoba (Argentina). Construct validity has been analyzed in study A using exploratory and confirmatory factor analysis. Furthermore, total-item correlation and internal consistency have been studied. Meanwhile, additional evidence was found in study B about the factorial structure and the convergent/discriminant validity in relation with the Positive and Negative Affective Scale. Moreover, internal consistency of the scale was also studied. In general terms, results indicate a unifactorial structure which explains 57.38% of the total variance, structure which has been replicated using confirmatory analysis in both samples. Internal consistency was acceptable in both studies (Cronbach's alpha above .68). In addition, higher scores of subjective happiness were positively and negatively related to positive affection and negative affection respectively. In conclusion, the results of these studies suggest that the scale is a valid and reliable measurement tool, allowing its continued use in future studies about subjective happiness.

Unexpected role for Helicobacter pylori DNA polymerase I as a source of genetic variability.

Helicobacter pylori, a human pathogen infecting about half of the world population, is characterised by its large intraspecies variability. Its genome plasticity has been invoked as the basis for its high adaptation capacity. Consistent with its small genome, H. pylori possesses only two bona fide DNA polymerases, Pol I and the replicative Pol III, lacking homologues of translesion synthesis DNA polymerases. Bacterial DNA polymerases I are implicated both in normal DNA replication and in DNA repair. We report that H. pylori DNA Pol I 5'- 3' exonuclease domain is essential for viability, probably through its involvement in DNA replication. We show here that, despite the fact that it also plays crucial roles in DNA repair, Pol I contributes to genomic instability. Indeed, strains defective in the DNA polymerase activity of the protein, although sensitive to genotoxic agents, display reduced mutation frequencies. Conversely, overexpression of Pol I leads to a hypermutator phenotype. Although the purified protein displays an intrinsic fidelity during replication of undamaged DNA, it lacks a proofreading activity, allowing it to efficiently elongate mismatched primers and perform mutagenic translesion synthesis. In agreement with this finding, we show that the spontaneous mutator phenotype of a strain deficient in the removal of oxidised pyrimidines from the genome is in part dependent on the presence of an active DNA Pol I. This study provides evidence for an unexpected role of DNA polymerase I in generating genomic plasticity.

Promoter DNA hypermethylation and gene repression in undifferentiated Arabidopsis cells.

Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.

The noncatalytic C-terminus of AtPOLK Y-family DNA polymerase affects synthesis fidelity, mismatch extension and translesion replication.

Cell survival depends not only on the ability to repair damaged DNA but also on the capability to perform DNA replication on unrepaired or imperfect templates. Crucial to this process are specialized DNA polymerases belonging to the Y family. These enzymes share a similar catalytic fold in their N-terminal region, and most of them have a less-well-conserved C-terminus which is not required for catalytic activity. Although this region is essential for appropriate localization and recruitment in vivo, its precise role during DNA synthesis remains unclear. Here we have compared the catalytic properties of AtPOLK, an Arabidopsis orthologue of mammalian pol kappa, and a truncated version lacking 193 amino acids from its C-terminus. We found that C-terminally truncated AtPOLK is a high-efficiency mutant protein, the DNA-binding capacity of which is not affected but it has higher catalytic efficiency and fidelity than the full-length enzyme. The truncated protein shows increased propensity to extend mispaired primer termini through misalignment and enhanced error-free bypass activity on DNA templates containing 7,8-dihydro-8-oxoGuanine. These results suggest that, in addition to facilitating recruitment to the replication fork, the C-terminus of Y-family DNA polymerases may also play a role in the kinetic control of their enzymatic activity.

Arabidopsis thaliana AtPOLK encodes a DinB-like DNA polymerase that extends mispaired primer termini and is highly expressed in a variety of tissues.

Cell survival after DNA damage depends on specialized DNA polymerases able to perform DNA synthesis on imperfect templates. Most of these enzymes belong to the recently discovered Y-family of DNA polymerases, none of which has been previously described in plants. We report here the isolation, functional characterization and expression analysis of a plant representative of the Y-family. This polymerase, which we have termed AtPolkappa, is a homolog of Escherichia coli pol IV and human pol kappa, and thus belongs to the DinB subfamily. We purified AtPolkappa and found a template-directed DNA polymerase, endowed with limited processivity that is able to extend primer-terminal mispairs. The activity and processivity of AtPolkappa are enhanced markedly upon deletion of 193 amino acids (aa) from its carboxy (C)-terminal domain. Loss of this region also affects the nucleotide selectivity of the enzyme, leading to the incorporation of both dCTP and dTTP opposite A in the template. We detected three cDNA forms, which result from the alternative splicing of AtPOLK mRNA and have distinct patterns of expression in different plant organs. Histochemical localization of beta-glucuronidase (GUS) activity in transgenic plants revealed that the AtPOLK promoter is active in endoreduplicating cells, suggesting a possible role during consecutive DNA replication cycles in the absence of mitosis.