1,720,962 research outputs found
Novel roles of DNA damage repair enzymes in the processing of modified ribonucleotides embedded in DNA
Full text linkFrom data in literature, it has well known that 100 million rNMPs are transiently present in mammalian DNA for several reasons. Moreover, the presence of RNA as single or more ribonucleotides into DNA results very dangerous for the cell because able to distort the double helix DNA. A specific pathway acts in order to remove this lesion called Ribonucleotide Excision Repair (RER) pathway in which RNase H2 has an important role as endonuclease, able to cleave at the 5’ side of rNMP in DNA. Although in last decade, huge steps forward have been done in this field, more studies are needed for better understanding the impact of this lesion on DNA and their back-up repair mechanism when RER does not work, as happens in several pathologies including cancer and Aicardi-Goutieres syndrome. Because of the high abundance of rNMPs in DNA and the ability of several polymerases to insert and elongate oxidized rGMP during DNA replication, the hypothesis of the incorporation of modified rNMPs (including abasic and oxidized rNMPs such as 7,8-dihydro-8-oxo-riboguanosine) results feasible. Moreover, these data underscore the possibility, not only regarding the presence of modified rNMPs into DNA, but also the necessity to determine how cells can target and remove oxidized or abasic rNMPs from DNA. Until now, nothing is known about the putative role of Base Excision Repair (BER) pathway in the removal of unmodified and modified rNMPs embedded in DNA. APE1 is the most important protein that works in BER pathway as endonuclease, able to cleave 5′ side of deoxyabasic sites generated spontaneously or following the processing of glycosylases (including OGG1) on a damaged (including oxidized) base. Moreover, APE1 has nucleotide incision repair activity on different modified bases, which are directly repaired by APE1 bypassing the action of specific glycosylases. Finally BER, but specifically APE1, has an important involvement in RNA metabolism and RNA-decay as demonstrated by its ability to cleave abasic single-stranded RNA and for having 3′-RNA phosphatase and weak 3′-5′ exoribonuclease activities. For this reason, there is a high likelihood that BER enzymes could be involved in the processing of rNMPs in DNA, particularly in the case of chemically modified rNMPs, such as abasic and oxidized rNMPs. Therefore, we focused our attention on enzymes belonging to RER and BER enzymes in order to investigate: If BER can be work as a back up repair mechanism when RER is inefficient; If RER is involved in the processing of abasic and oxidized rNMPs embedded in DNA; and if not, if BER is involved in the processing of abasic and oxidized rNMPs embedded in DNA. For all the experiments, we have used different DNA oligonucleotides in which an abasic or an oxidized ribonucleotide was embedded in the middle of the sequence. First, we tested recombinant and cell-free extracts RNase H2. Surprisingly, we discovered that RNase H2, although able to process normal rG embedded in DNA, is not able to cleave abasic and oxidized rNMPs embedded in DNA leaving the hypothesis that RER can not work on this lesion. After that, we moved on BER pathway, specifically on APE1 and OGG1 proteins. We discovered that APE1, as 5’-endonuclease, does not cleave unmodified rNMPs embedded in DNA. So, APE1 may not work as back up when RNase H2 is inefficient as happens for different pathologies including cancer and Aicardi-Goutieres syndrome. We discovered and characterized an unknown APE1 activity on abasic ribonucleotide embedded in DNA. We then analyzed the activities of 8-oxoguanine DNA glycosylase (OGG1) and APE1 to recognize and cleave an oxidized rNMP. Our data demonstrate that OGG1, although able to bind the oxidized rG, is inefficient in the cleavage of this substrate. Surprisingly, APE1 shows a weak endoribonuclease activity on the oxidized substrate, maybe associable to a putative NIR activity of APE1. All these results show a strong impact on the DNA repair field.From data in literature, it has well known that 100 million rNMPs are transiently present in mammalian DNA for several reasons. Moreover, the presence of RNA as single or more ribonucleotides into DNA results very dangerous for the cell because able to distort the double helix DNA. A specific pathway acts in order to remove this lesion called Ribonucleotide Excision Repair (RER) pathway in which RNase H2 has an important role as endonuclease, able to cleave at the 5’ side of rNMP in DNA. Although in last decade, huge steps forward have been done in this field, more studies are needed for better understanding the impact of this lesion on DNA and their back-up repair mechanism when RER does not work, as happens in several pathologies including cancer and Aicardi-Goutieres syndrome. Because of the high abundance of rNMPs in DNA and the ability of several polymerases to insert and elongate oxidized rGMP during DNA replication, the hypothesis of the incorporation of modified rNMPs (including abasic and oxidized rNMPs such as 7,8-dihydro-8-oxo-riboguanosine) results feasible. Moreover, these data underscore the possibility, not only regarding the presence of modified rNMPs into DNA, but also the necessity to determine how cells can target and remove oxidized or abasic rNMPs from DNA. Until now, nothing is known about the putative role of Base Excision Repair (BER) pathway in the removal of unmodified and modified rNMPs embedded in DNA. APE1 is the most important protein that works in BER pathway as endonuclease, able to cleave 5′ side of deoxyabasic sites generated spontaneously or following the processing of glycosylases (including OGG1) on a damaged (including oxidized) base. Moreover, APE1 has nucleotide incision repair activity on different modified bases, which are directly repaired by APE1 bypassing the action of specific glycosylases. Finally BER, but specifically APE1, has an important involvement in RNA metabolism and RNA-decay as demonstrated by its ability to cleave abasic single-stranded RNA and for having 3′-RNA phosphatase and weak 3′-5′ exoribonuclease activities. For this reason, there is a high likelihood that BER enzymes could be involved in the processing of rNMPs in DNA, particularly in the case of chemically modified rNMPs, such as abasic and oxidized rNMPs. Therefore, we focused our attention on enzymes belonging to RER and BER enzymes in order to investigate: If BER can be work as a back up repair mechanism when RER is inefficient; If RER is involved in the processing of abasic and oxidized rNMPs embedded in DNA; and if not, if BER is involved in the processing of abasic and oxidized rNMPs embedded in DNA. For all the experiments, we have used different DNA oligonucleotides in which an abasic or an oxidized ribonucleotide was embedded in the middle of the sequence. First, we tested recombinant and cell-free extracts RNase H2. Surprisingly, we discovered that RNase H2, although able to process normal rG embedded in DNA, is not able to cleave abasic and oxidized rNMPs embedded in DNA leaving the hypothesis that RER can not work on this lesion. After that, we moved on BER pathway, specifically on APE1 and OGG1 proteins. We discovered that APE1, as 5’-endonuclease, does not cleave unmodified rNMPs embedded in DNA. So, APE1 may not work as back up when RNase H2 is inefficient as happens for different pathologies including cancer and Aicardi-Goutieres syndrome. We discovered and characterized an unknown APE1 activity on abasic ribonucleotide embedded in DNA. We then analyzed the activities of 8-oxoguanine DNA glycosylase (OGG1) and APE1 to recognize and cleave an oxidized rNMP. Our data demonstrate that OGG1, although able to bind the oxidized rG, is inefficient in the cleavage of this substrate. Surprisingly, APE1 shows a weak endoribonuclease activity on the oxidized substrate, maybe associable to a putative NIR activity of APE1. All these results show a strong impact on the DNA repair field
Unveiling the non-repair face of the Base Excision Repair pathway in RNA processing: A missing link between DNA repair and gene expression?
Full text linkHuman AP-endonuclease (Ape1) activity on telomeric G4 structures is modulated by acetylatable lysine residues in the N-terminal sequence
No full textLoss of telomeres stability is a hallmark of cancer cells. Exposed telomeres are prone to aberrant end joining reactions leading to chromosomal fusions and translocations. Human telomeres contain repeated TTAGGG elements, in which the 3' exposed strand may adopt a G-quadruplex (G4) structure. The guanine-rich regions of telomeres are hotspots for oxidation forming 8-oxoguanine, a lesion that is handled by the base excision repair (BER) pathway. One key player of this pathway is Ape1, the main human endonuclease processing abasic sites. Recent evidences showed an important role for Ape1 in telomeric physiology, but the molecular details regulating Ape1 enzymatic activities on G4-telomeric sequences are lacking. Through a combination of in vitro assays, we demonstrate that Ape1 can bind and process different G4 structures and that this interaction involves specific acetylatable lysine residues (i.e. K-27/31/32/35) present in the unstructured N-terminal sequence of the protein. The cleavage of an abasic site located in a G4 structure by Ape1 depends on the DNA conformation or the position of the lesion and on electrostatic interactions between the protein and the nucleic acids. Moreover, Ape1 mutants mimicking the acetylated protein display increased cleavage activity for abasic sites. We found that nucleophosmin (NPM1), which binds the N-terminal sequence of Ape1, plays a role in modulating telomere length and Ape1 activity at abasic G4 structures. Thus, the Ape1 N-terminal sequence is an important relay site for regulating the enzyme's activity on G4-telomeric sequences, and specific acetylatable lysine residues constitute key regulatory sites of Ape1 enzymatic activity dynamics at telomeres
Platinum Salts in Patients with Breast Cancer: A Focus on Predictive Factors
Full text linkBreast cancer (BC) is the most frequent oncologic cause of death among women and the improvement of its treatments is compelling. Platinum salts (e.g., carboplatin, cisplatin, and oxaliplatin) are old drugs still used to treat BC, especially the triple-negative subgroup. However, only a subset of patients see a concrete benefit from these drugs, raising the question of how to select them properly. Therefore, predictive biomarkers for platinum salts in BC still represent an unmet clinical need. Here, we review clinical and preclinical works in order to summarize the current evidence about predictive or putative platinum salt biomarkers in BC. The association between BRCA1/2 gene mutations and platinum sensitivity has been largely described. However, beyond the mutations of these two genes, several other proteins belonging to the homologous recombination pathways have been linked to platinum response, defining the concept of BRCAness. Several works, here reviewed, have tried to capture BRCAness through different strategies, such as homologous recombination deficiency (HRD) score and genetic signatures. Moreover, p53 and its family members (p63 and p73) might also be used as predictors of platinum response. Finally, we describe the mounting preclinical evidence regarding base excision repair deficiency as a possible new platinum biomarker
Expression of APE1 endonuclease in primarily cultured aortic valve interstitial cells undergoing spontaneous senescence
No full textApurinic/apyrimidinic endodeoxyribonuclease 1
(APE1) is the major enzyme involved in the base excision
repair pathway working on DNA damages mainly
caused by oxidative stress. Namely, APE1 hydrolyses
the phosphodiester bond at the abasic sites generated
by DNA glycosylases, creating the substrate for DNA
polymerase β and DNA ligase IIIa which terminate the
reparative process [1]. APE1 also seems to play a role in
cell senescence maintaining telomere stability and size
in interaction with specifi c telomere-protective proteins
[2]. Here, aortic valve interstitial cells (AVICs) isolated
from healthy bovine valve leafl ets were cultured under
normal conditions for up to 90 days to achieve spontaneous
cell senescence. Time-dependent increase in
β-galactosidase activity, a marker of cell senescence, was
paralleled by a remarkable decrease of APE1-expressing
AVICs starting from day 60, as immunocytochemically
revealed. Quantitative Western blot analyses also showed
a drop of APE1 protein content at day 60, whereas RTPCR
analyses revealed a mild increase of the enzyme
mRNA over time. Ultrastructurally, AVICs appeared
well preserved up to 30-day-long culturing. Conversely,
starting from day 60, cells showed non-lysosomal
autophagocytosis features mainly consisting of a hypertrophic
rough endoplasmic reticulum engulfi ng suff ering
mitochondria [3]. Cytoplasm vacuolization due to
large organelle degeneration was also clearly appreciable.
In conclusion, decreasing APE1 expression over time
is supposed to contribute to AVIC decay in a model of
spontaneous cell senescence
Inhibitors of the apurinic/apyrimidinic endonuclease 1 (APE1)/nucleophosmin (NPM1) interaction that display anti-tumor properties
No full textThe apurinic/apyrimidinic endonuclease 1 (APE1) is a protein central to the base excision DNA repair pathway and operates in the modulation of gene expression through redox-dependent and independent mechanisms. Aberrant expression and localization of APE1 in tumors are recurrent hallmarks of aggressiveness and resistance to therapy. We identified and characterized the molecular association between APE1 and nucleophosmin (NPM1), a multifunctional protein involved in the preservation of genome stability and rRNA maturation. This protein-protein interaction modulates subcellular localization and endonuclease activity of APE1. Moreover, we reported a correlation between APE1 and NPM1 expression levels in ovarian cancer, with NPM1 overexpression being a marker of poor prognosis. These observations suggest that tumors that display an augmented APE1/NPM1 association may exhibit increased aggressiveness and resistance. Therefore, targeting the APE1/NPM1 interaction might represent an innovative strategy for the development of anticancer drugs, as tumor cells relying on higher levels of APE1 and NPM1 for proliferation and survival may be more sensitive than untransformed cells. We set up a chemiluminescence-based high-throughput screening assay in order to find small molecules able to interfere with the APE1/NPM1 interaction. This screening led to the identification of a set of bioactive compounds that impair the APE1/NPM1 association in living cells. Interestingly, some of these molecules display anti-proliferative activity and sensitize cells to therapeutically relevant genotoxins. Given the prognostic significance of APE1 and NPM1, these compounds might prove effective in the treatment of tumors that show abundant levels of both proteins, such as ovarian or hepatic carcinomas. © 2015 Wiley Periodicals, Inc
Inhibition of APE1-endonuclease activity affects cell metabolism in colon cancer cells via a p53-dependent pathway
Full text linkThe pathogenesis of colorectal cancer (CRC) involves different mechanisms, such as genomic and microsatellite instabilities. Recently, a contribution of the base excision repair (BER) pathway in CRC pathology has been emerged. In this context, the involvement of APE1 in the BER pathway and in the transcriptional regulation of genes implicated in tumor progression strongly correlates with chemoresistance in CRC and in more aggressive cancers. In addition, the APE1 interactome is emerging as an important player in tumor progression, as demonstrated by its interaction with Nucleophosmin (NPM1). For these reasons, APE1 is becoming a promising target in cancer therapy and a powerful prognostic and predictive factor in several cancer types. Thus, specific APE1 inhibitors have been developed targeting: i) the endonuclease activity; ii) the redox function and iii) the APE1-NPM1 interaction. Furthermore, mutated p53 is a common feature of advanced CRC. The relationship between APE1 inhibition and p53 is still completely unknown. Here, we demonstrated that the inhibition of the endonuclease activity of APE1 triggers p53-mediated effects on cell metabolism in HCT-116 colon cancer cell line. In particular, the inhibition of the endonuclease activity, but not of the redox function or of the interaction with NPM1, promotes p53 activation in parallel to sensitization of p53-expressing HCT-116 cell line to genotoxic treatment. Moreover, the endonuclease inhibitor affects mitochondrial activity in a p53-dependent manner. Finally, we demonstrated that 3D organoids derived from CRC patients are susceptible to APE1-endonuclease inhibition in a p53-status correlated manner, recapitulating data obtained with HCT-116 isogenic cell lines. These findings suggest the importance of further studies aimed at testing the possibility to target the endonuclease activity of APE1 in CRC
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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