18 research outputs found
Demethylation initiated by ROS1 glycosylase involves random sliding along DNA
Active DNA demethylation processes play a critical
role in shaping methylation patterns, yet our
understanding of the mechanisms involved is still
fragmented and incomplete. REPRESSOR OF
SILENCING 1 (ROS1) is a prototype member of a
family of plant 5-methylcytosine DNA glycosylases
that initiate active DNA demethylation through a
base excision repair pathway. As ROS1 binds DNA
non-specifically, we have critically tested the hypothesis
that facilitated diffusion along DNA may
contribute to target location by the enzyme. We
have found that dissociation of ROS1 from DNA is
severely restricted when access to both ends is obstructed
by tetraloops obstacles. Unblocking any
end facilitates protein dissociation, suggesting that
random surface sliding is the main route to a
specific target site. We also found that removal of
the basic N-terminal domain of ROS1 significantly
impairs the sliding capacity of the protein. Finally,
we show that sliding increases the catalytic efficiency
of ROS1 on 5-meC:G pairs, but not on T:G
mispairs, thus suggesting that the enzyme
achieves recognition and excision of its two substrate
bases by different means. A model is
proposed to explain how ROS1 finds its potential
targets on DNA
Bases moleculares de la desmetilación de DNA en Arabidopsis thaliana
Resumen de la comunicación presentada al XX Congreso de la Sociedad Española de Mutagénesis Ambiental – Córdoba 201
Early steps of active DNA demethylation initiated by ROS1 glycosylase require three putative helix-invading residues
Active DNA demethylation is crucial for epigenetic control, but the underlying enzymatic mechanisms are incompletely understood. REPRESSOR OF SILENCING 1 (ROS1) is a 5-methylcytosine (5-meC) DNA glycosylase/lyase that initiates DNA demethylation in plants through a base excision repair process. The enzyme binds DNA nonspecifically and slides along the substrate in search of 5-meC. In this work, we have used homology modelling and biochemical analysis to gain insight into the mechanism of target location and recognition by ROS1. We have found that three putative helix-intercalating residues (Q607, R903 and M905) are required for processing of 5-meC:G pairs, but dispensable for excision of mismatched 5-meC. Mutant proteins Q607A, R903A and M905G retain the capacity to process an abasic site opposite G, thus suggesting that all three residues play a critical role in early steps of the base extrusion process and likely contribute to destabilization of 5-meC:G pairs. While R903 and M905 are not essential for DNA binding, mutation of Q607 abrogates stable binding to both methylated and nonmethylated DNA. However, the mutant protein Q607A can form stable complexes with DNA substrates containing blocked ends, which suggests that Q607 intercalates into the helix and inhibits sliding. Altogether, our results suggest that ROS1 uses three predicted helix-invading residues to actively interrogate DNA in search for 5-meC
Desmetilación activa del ADN: un mecanismo epigenético para la reactivación de genes silenciados
Los mecanismos de control epigenético son esenciales para una regulación estable de los patrones de
expresión génica y desempeñan un papel central en los ciclos de vida de animales y plantas. La metilación de
la citosina en el carbono 5 del anillo de pirimidina (5-meC) es una marca epigenética estable, pero reversible,
que promueve el silenciamiento génico transcripcional. Comprender cómo se regula el estado de metilación
del genoma a nivel global o local requiere una definición de los procesos enzimáticos que metilan y
desmetilan el ADN. Sin embargo, aunque las enzimas responsables del establecimiento y mantenimiento de la
metilación de ADN han sido bien caracterizadas, los mecanismos de desmetilación no se conocen con
exactitud. Nuestro grupo, junto con otros, ha obtenido datos genéticos y bioquímicos que sugieren que dos
proteínas de Arabidopsis con dominio ADN glicosilasa (ROS1 y DME) actúan como ADN desmetilasas
capaces de activar la expresión de genes previamente silenciados. Nuestros resultados previos indican que
ROS1 y DME catalizan la liberación de 5-meC del ADN mediante un mecanismo ADN glicosilasa. Estos
resultados sugieren que una de las funciones de ROS1 y DME es iniciar el borrado de 5-meC mediante un
proceso de escisión de bases y proporcionan una importante evidencia bioquímica a favor de la existencia de
una ruta de desmetilación activa en plantas. En la actualidad, nuestro grupo de investigación se concentra en
caracterizar funcionalmente este novedoso mecanismo de control epigenético mediante una aproximación
multidisciplinar que combina metodologías del campo de la bioquímica, la genética y la biofísica. Este estudio
suministrará una información esencial para entender los mecanismos responsables de la reprogramación
epigenética en el núcleo celular, con aplicaciones potenciales en biotecnología y biomedicin
A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine
DNA cytosine methylation (5-meC) is a widespread epigenetic mark associated to gene silencing. In plants, DEMETER-LIKE (DML) proteins typified by Arabidopsis REPRESSOR OF SILENCING 1 (ROS1) initiate active DNA demethylation by catalyzing 5-meC excision. DML proteins belong to the HhH-GPD superfamily, the largest and most functionally diverse group of DNA glycosylases, but the molecular properties that underlie their capacity to specifically recognize and excise 5-meC are largely unknown. We have found that sequence similarity to HhH-GPD enzymes in DML proteins is actually distributed over two non-contiguous segments connected by a predicted disordered region. We used homology-based modeling to locate candidate residues important for ROS1 function in both segments, and tested our predictions by site-specific mutagenesis. We found that amino acids T606 and D611 are essential for ROS1 DNA glycosylase activity, whereas mutations in either of two aromatic residues (F589 and Y1028) reverse the characteristic ROS1 preference for 5-meC over T. We also found evidence suggesting that ROS1 uses Q607 to flip out 5-meC, while the contiguous N608 residue contributes to sequence-context specificity. In addition to providing novel insights into the molecular basis of 5-meC excision, our results reveal that ROS1 and its DML homologs possess a discontinuous catalytic domain that is unprecedented among known DNA glycosylases
ROS1 5-methylcytosine DNA glycosylase is a slow-turnover catalyst that initiates DNA demethylation in a distributive fashion
Arabidopsis ROS1 belongs to a family of plant 5-methycytosine DNA glycosylases that initiate DNA demethylation through base excision. ROS1 displays the remarkable capacity to excise 5-meC, and to a lesser extent T, while retaining the ability to discriminate effectively against C and U. We found that replacement of the C5-methyl group by halogen substituents greatly decreased excision of the target base. Furthermore, 5-meC was excised more efficiently from mismatches, whereas excision of T only occurred when mispaired with G. These results suggest that ROS1 specificity arises by a combination of selective recognition at the active site and thermodynamic stability of the target base. We also found that ROS1 is a low-turnover catalyst because it binds tightly to the abasic site left after 5-meC removal. This binding leads to a highly distributive behaviour of the enzyme on DNA substrates containing multiple 5-meC residues, and may help to avoid generation of double-strand breaks during processing of bimethylated CG dinucleotides. We conclude that the biochemical properties of ROS1 are consistent with its proposed role in protecting the plant genome from excess methylation
Correction : Chaparro et al. Incidence, Clinical Characteristics and Management of Inflammatory Bowel Disease in Spain: Large-Scale Epidemiological Study. J. Clin. Med. 2021, 10, 2885
The authors wish to make the following corrections to this paper [...]
Incidence, Clinical Characteristics and Management of Inflammatory Bowel Disease in Spain : Large-Scale Epidemiological Study
(1) Aims: To assess the incidence of inflammatory bowel disease (IBD) in Spain, to describe the main epidemiological and clinical characteristics at diagnosis and the evolution of the disease, and to explore the use of drug treatments. (2) Methods: Prospective, population-based nationwide registry. Adult patients diagnosed with IBD-Crohn's disease (CD), ulcerative colitis (UC) or IBD unclassified (IBD-U)-during 2017 in Spain were included and were followed-up for 1 year. (3) Results: We identified 3611 incident cases of IBD diagnosed during 2017 in 108 hospitals covering over 22 million inhabitants. The overall incidence (cases/100,000 person-years) was 16 for IBD, 7.5 for CD, 8 for UC, and 0.5 for IBD-U; 53% of patients were male and median age was 43 years (interquartile range = 31-56 years). During a median 12-month follow-up, 34% of patients were treated with systemic steroids, 25% with immunomodulators, 15% with biologics and 5.6% underwent surgery. The percentage of patients under these treatments was significantly higher in CD than UC and IBD-U. Use of systemic steroids and biologics was significantly higher in hospitals with high resources. In total, 28% of patients were hospitalized (35% CD and 22% UC patients, p < 0.01). (4) Conclusion: The incidence of IBD in Spain is rather high and similar to that reported in Northern Europe. IBD patients require substantial therapeutic resources, which are greater in CD and in hospitals with high resources, and much higher than previously reported. One third of patients are hospitalized in the first year after diagnosis and a relevant proportion undergo surgery
Risk Factors for COVID-19 in Inflammatory Bowel Disease: A National, ENEIDA-Based Case–Control Study (COVID-19-EII)
(1) Scant information is available concerning the characteristics that may favour the acquisition of COVID-19 in patients with inflammatory bowel disease (IBD). Therefore, the aim of this study was to assess these differences between infected and noninfected patients with IBD. (2) This nationwide case-control study evaluated patients with inflammatory bowel disease with COVID-19 (cases) and without COVID-19 (controls) during the period March-July 2020 included in the ENEIDA of GETECCU. (3) A total of 496 cases and 964 controls from 73 Spanish centres were included. No differences were found in the basal characteristics between cases and controls. Cases had higher comorbidity Charlson scores (24% vs. 19%; p = 0.02) and occupational risk (28% vs. 10.5%; p < 0.0001) more frequently than did controls. Lockdown was the only protective measure against COVID-19 (50% vs. 70%; p < 0.0001). No differences were found in the use of systemic steroids, immunosuppressants or biologics between cases and controls. Cases were more often treated with 5-aminosalicylates (42% vs. 34%; p = 0.003). Having a moderate Charlson score (OR: 2.7; 95%CI: 1.3-5.9), occupational risk (OR: 2.9; 95%CI: 1.8-4.4) and the use of 5-aminosalicylates (OR: 1.7; 95%CI: 1.2-2.5) were factors for COVID-19. The strict lockdown was the only protective factor (OR: 0.1; 95%CI: 0.09-0.2). (4) Comorbidities and occupational exposure are the most relevant factors for COVID-19 in patients with IBD. The risk of COVID-19 seems not to be increased by immunosuppressants or biologics, with a potential effect of 5-aminosalicylates, which should be investigated further and interpreted with caution