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1.
Nat Commun ; 11(1): 4826, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958757

RESUMO

DNA replication initiates from multiple genomic locations called replication origins. In metazoa, DNA sequence elements involved in origin specification remain elusive. Here, we examine pluripotent, primary, differentiating, and immortalized human cells, and demonstrate that a class of origins, termed core origins, is shared by different cell types and host ~80% of all DNA replication initiation events in any cell population. We detect a shared G-rich DNA sequence signature that coincides with most core origins in both human and mouse genomes. Transcription and G-rich elements can independently associate with replication origin activity. Computational algorithms show that core origins can be predicted, based solely on DNA sequence patterns but not on consensus motifs. Our results demonstrate that, despite an attributed stochasticity, core origins are chosen from a limited pool of genomic regions. Immortalization through oncogenic gene expression, but not normal cellular differentiation, results in increased stochastic firing from heterochromatin and decreased origin density at TAD borders.


Assuntos
DNA/biossíntese , DNA/química , Origem de Replicação/genética , Animais , Composição de Bases , Sequência de Bases , Carcinogênese , Diferenciação Celular , Células Cultivadas , Replicação do DNA/genética , Genoma Humano/genética , Heterocromatina/genética , Humanos , Camundongos , Motivos de Nucleotídeos , Transcrição Genética
2.
BMC Evol Biol ; 20(1): 75, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32590933

RESUMO

BACKGROUND: We hypothesize prebiotic evolution of self-replicating macro-molecules (Alberts, Molecular biology of the cell, 2015; Orgel, Crit Rev Biochem Mol Biol 39:99-123, 2004; Hud, Nat Commun 9:5171) favoured the constituent nucleotides and biophysical properties observed in the RNA and DNA of modern organisms. Assumed initial conditions are a shallow tide pool, containing a racemic mix of diverse nucleotide monomers (Barks et al., Chembiochem 11:1240-1243, 2010; Krishnamurthy, Nat Commun 9:5175, 2018; Hirao, Curr Opin Chem Biol 10:622-627), subject to day/night thermal fluctuations (Piccirilli et al., Nature 343:33-37, 1990). Self-replication, like Polymerase Chain Reactions, followed as higher daytime thermal energy "melted" inter-strand hydrogen bonds causing strand separation while solar UV radiation increased prebiotic nucleobase formation (Szathmary, Proc Biol Sci 245:91-99, 1991; Materese et al., Astrobiology 17:761-770, 2017; Bera et al., Astrobiology 17:771-785, 2017). Lower night energies allowed free monomers to form hydrogen bonds with their template counterparts leading to daughter strand synthesis (Hirao, Biotechniques 40:711, 2006). RESULTS: Evolutionary selection favoured increasing strand length to maximize auto-catalytic function in RNA and polymer stability in double stranded DNA (Krishnamurthy, Chemistry 24:16708-16715, 2018; Szathmary, Nat Rev Genet 4:995-1001, 2003). However, synthesis of the full daughter strand before daytime temperatures produced strand separation, longer polymer length required increased speed of self-replication. Computer simulations demonstrate optimal polynucleotide autocatalytic speed is achieved when the constituent nucleotides possess a left-right asymmetry that decreases the hydrogen bond kinetic barrier for the free nucleotide attachment to the template on one side and increases bond barrier on the other side preventing it from releasing prior to covalent bond formation. This phenomenon is similar to asymmetric kinetics observed during polymerization of the front and the back ends of linear cytoskeletal proteins such as actin and microtubules (Orgel, Nature 343:18-20, 1990; Henry, Curr Opin Chem Biol 7:727-733, 2003; Walker et al., J Cell Biol 108:931-937, 1989; Crevenna et al., J Biol Chem 288:12102-12113, 2013). Since rotation of the nucleotide would disrupt the asymmetry, the optimal nucleotides must form two or more hydrogen bonds with their counterpart on the template strand. All nucleotides in modern RNA and DNA have these predicted properties. Our models demonstrate these constraints on the properties of constituent monomers result in biophysical properties found in modern DNA and RNA including strand directionality, anti-parallel strand orientation, homochirality, quadruplet alphabet, and complementary base pairing. Furthermore, competition between RNA and DNA auto-replicators for 3 nucleotides in common permit states coexistence and possible cooperative interactions that could be incorporated into nascent living systems. CONCLUSION: Our findings demonstrate the molecular properties of DNA/RNA could have emerged from Darwinian competition among macromolecular replicators that selected nucleotide monomers that maximized the speed of autocatalysis.


Assuntos
Replicação do DNA , DNA/biossíntese , Polinucleotídeos/biossíntese , Prebióticos , RNA/biossíntese , DNA/genética , Cinética , Polinucleotídeos/genética , RNA/genética
3.
Adv Exp Med Biol ; 1241: 35-45, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32383114

RESUMO

Polymerase δ-interacting protein 2 (PolDIP2) is involved in the multiple protein-protein interactions and plays roles in many cellular processes including regulation of the nuclear redox environment, organization of the mitotic spindle and chromosome segregation, pre-mRNA processing, mitochondrial morphology and functions, cell migration and cellular adhesion. PolDIP2 is also a binding partner of high-fidelity DNA polymerase delta, PCNA and a number of translesion and repair DNA polymerases. The growing evidence suggests that PolDIP2 is a general regulatory protein in DNA damage response. However PolDIP2 functions in DNA translesion synthesis and repair are not fully understood. In this review, we address the functional interaction of PolDIP2 with human DNA polymerases and discuss the possible functions in DNA damage response.


Assuntos
Dano ao DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , Proteínas Nucleares/metabolismo , Humanos , Antígeno Nuclear de Célula em Proliferação/metabolismo
4.
Mol Cell ; 78(6): 1237-1251.e7, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32442397

RESUMO

DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy.


Assuntos
Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , DNA/biossíntese , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , DNA/genética , Dano ao DNA/genética , DNA Primase/metabolismo , DNA Primase/fisiologia , Reparo do DNA/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/fisiologia , Células HEK293 , Humanos , Células K562 , Enzimas Multifuncionais/metabolismo , Enzimas Multifuncionais/fisiologia , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/fisiologia , Fatores de Transcrição/genética
5.
PLoS One ; 15(4): e0227849, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32343690

RESUMO

Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase.


Assuntos
Quebras de DNA/efeitos da radiação , Reparo do DNA , DNA/biossíntese , Fase G1/genética , Mitose/genética , Animais , Linhagem Celular , DNA/genética , DNA/efeitos da radiação , Fase G1/efeitos da radiação , Humanos , Raios Infravermelhos/efeitos adversos , Lasers/efeitos adversos , Mitose/efeitos da radiação , Potoroidae
6.
Am J Physiol Lung Cell Mol Physiol ; 318(6): L1165-L1171, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32292070

RESUMO

Bronchopulmonary dysplasia (BPD), a long-term respiratory morbidity of prematurity, is characterized by attenuated alveolar and vascular development. Supplemental oxygen and immature antioxidant defenses contribute to BPD development. Our group identified thioredoxin reductase-1 (TXNRD1) as a therapeutic target to prevent BPD. The present studies evaluated the impact of the TXNRD1 inhibitor aurothioglucose (ATG) on pulmonary responses and gene expression in newborn C57BL/6 pups treated with saline or ATG (25 mg/kg ip) within 12 h of birth and exposed to room air (21% O2) or hyperoxia (>95% O2) for 72 h. Purified RNA from lung tissues was sequenced, and differential expression was evaluated. Hyperoxic exposure altered ~2,000 genes, including pathways involved in glutathione metabolism, intrinsic apoptosis signaling, and cell cycle regulation. The isolated effect of ATG treatment was limited primarily to genes that regulate angiogenesis and vascularization. In separate studies, pups were treated as described above and returned to room air until 14 days. Vascular density analyses were performed, and ANOVA indicated an independent effect of hyperoxia on vascular density and alveolar architecture at 14 days. Consistent with RNA-seq analyses, ATG significantly increased vascular density in room air, but not in hyperoxia-exposed pups. These findings provide insights into the mechanisms by which TXNRD1 inhibitors may enhance lung development.


Assuntos
Ar , Aurotioglucose/farmacologia , Hiperóxia/patologia , Pulmão/irrigação sanguínea , Pulmão/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Doença Aguda , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Apoptose/genética , DNA/biossíntese , Glutationa/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/embriologia , Camundongos Endogâmicos C57BL , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/embriologia , Alvéolos Pulmonares/patologia , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Transcriptoma/genética , Regulação para Cima/efeitos dos fármacos
7.
Nucleic Acids Res ; 48(8): 4298-4308, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32182354

RESUMO

The RNF168 E3 ubiquitin ligase is activated in response to double stranded DNA breaks (DSBs) where it mono-ubiquitinates γH2AX (ub-H2AX). RNF168 protein expression and ubiquitin signaling are finely regulated during the sensing, repair and resolution of DNA damage in order to avoid excessive spreading of ubiquitinated chromatin. Supra-physiological RNF168 protein expression levels have been shown to block DNA end resection at DSBs and increase PARP inhibitor (PARPi) sensitivity. In this study, we examined the impact of ectopic RNF168 overexpression on hydroxyurea (HU)-induced stalled replication forks in the setting of BRCA1 deficiency. Surprisingly, RNF168 overexpression resulted in the extension of DNA fibers, despite the presence of HU, in BRCA1 deficient cells. Mechanistically, RNF168 overexpression recruited RAD18 to ub-H2AX at HU-induced DNA breaks. Subsequently, a RAD18-SLF1 axis was responsible for initiating DNA synthesis in a manner that also required the break-induced replication (BIR) factors RAD52 and POLD3. Strikingly, the presence of wild-type BRCA1 blocked RNF168-induced DNA synthesis. Notably, BIR-like repair has previously been linked with tandem duplication events found in BRCA1-mutated genomes. Thus, in the absence of BRCA1, excessive RNF168 expression may drive BIR, and contribute to the mutational signatures observed in BRCA1-mutated cancers.


Assuntos
Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA/biossíntese , Ubiquitina-Proteína Ligases/metabolismo , Proteína BRCA1/genética , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Hidroxiureia/farmacologia , Reparo de DNA por Recombinação , Ubiquitinação
9.
Elife ; 92020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31934863

RESUMO

Telomeres are a significant challenge to DNA replication and are prone to replication stress and telomere fragility. The shelterin component TRF1 facilitates telomere replication but the molecular mechanism remains uncertain. By interrogating the proteomic composition of telomeres, we show that mouse telomeres lacking TRF1 undergo protein composition reorganisation associated with the recruitment of DNA damage response and chromatin remodellers. Surprisingly, mTRF1 suppresses the accumulation of promyelocytic leukemia (PML) protein, BRCA1 and the SMC5/6 complex at telomeres, which is associated with increased Homologous Recombination (HR) and TERRA transcription. We uncovered a previously unappreciated role for mTRF1 in the suppression of telomere recombination, dependent on SMC5 and also POLD3 dependent Break Induced Replication at telomeres. We propose that TRF1 facilitates S-phase telomeric DNA synthesis to prevent illegitimate mitotic DNA recombination and chromatin rearrangement.


Assuntos
Montagem e Desmontagem da Cromatina , Quebras de DNA , Replicação do DNA/genética , Recombinação Genética/genética , Telômero/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatina/metabolismo , DNA/biossíntese , DNA Polimerase III/metabolismo , Deleção de Genes , Humanos , Corpos de Inclusão/metabolismo , Camundongos , Mitose , Regulação para Cima/genética
10.
Methods Mol Biol ; 2102: 483-507, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989573

RESUMO

The unscheduled DNA synthesis (UDS) assay measures the ability of a cell to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique by creating 6-4 photoproducts and pyrimidine dimers using UV-C (254 nm) irradiation. This procedure is designed specifically for quantification of the 6-4 photoproducts. Repair is quantified by the amount of radioactive thymidine incorporated during repair synthesis after this insult, and radioactivity is evaluated by grain counting after autoradiography. The results have been used to clinically diagnose human DNA repair deficiency disorders, and provide a basis for investigation of repair deficiency in human tissues or tumors. Genomic sequencing to establish the presence of specific mutations is also used now for clinical diagnosis of DNA repair deficiency syndromes. Few functional assays are available which directly measure the capacity to perform NER on the entire genome. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation (HCR). As discussed in Chap. 28 is not an equivalent technique, as it measures only transcription-coupled repair (TCR) at active genes, a small subset of total NER. Our laboratory also explored the fluorescent label-based Click-iT assay that uses EdU as the label, rather than 3H thymidine. Despite emerging studies in the literature finding this assay to be useful for other purposes, we found that the EdU-based UDS assay was not consistent or reproducible compared with the 3H thymidine-based assay.


Assuntos
Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , DNA/biossíntese , Dímeros de Pirimidina/efeitos da radiação , Células Cultivadas , DNA/efeitos da radiação , Dano ao DNA/genética , Reparo do DNA/efeitos da radiação , Nucleotídeos de Desoxiuracil , Técnicas Genéticas/instrumentação , Genômica , Humanos , Dímeros de Pirimidina/genética , Timidina , Trítio , Raios Ultravioleta , Fluxo de Trabalho
12.
Nature ; 575(7783): 523-527, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31723267

RESUMO

The protection of telomere ends by the shelterin complex prevents DNA damage signalling and promiscuous repair at chromosome ends. Evidence suggests that the 3' single-stranded telomere end can assemble into a lasso-like t-loop configuration1,2, which has been proposed to safeguard chromosome ends from being recognized as DNA double-strand breaks2. Mechanisms must also exist to transiently disassemble t-loops to allow accurate telomere replication and to permit telomerase access to the 3' end to solve the end-replication problem. However, the regulation and physiological importance of t-loops in the protection of telomere ends remains unknown. Here we identify a CDK phosphorylation site in the shelterin subunit at Ser365 of TRF2, whose dephosphorylation in S phase by the PP6R3 phosphatase provides a narrow window during which the RTEL1 helicase can transiently access and unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 at Ser365 outside of S phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate activation of ATM, but also counteracts replication conflicts at DNA secondary structures that arise within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates the assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response.


Assuntos
Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Fosfosserina/metabolismo , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , DNA/biossíntese , DNA/química , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , Fibroblastos , Genoma/genética , Células HEK293 , Humanos , Camundongos , Mutação , Fenótipo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fase S , Telomerase/metabolismo , Telômero/genética , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/genética
13.
PLoS Genet ; 15(11): e1008426, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31765372

RESUMO

DNA polymerase epsilon (Pol ε) is critical for genome duplication, but little is known about how post-translational modification regulates its function. Here we report that the Pol ε catalytic subunit Pol2 in yeast is sumoylated at a single lysine within a catalytic domain insertion uniquely possessed by Pol2 family members. We found that Pol2 sumoylation occurs specifically in S phase and is increased under conditions of replication fork blockade. Analyses of the genetic requirements of this modification indicate that Pol2 sumoylation is associated with replication fork progression and dependent on the Smc5/6 SUMO ligase known to promote DNA synthesis. Consistently, the pol2 sumoylation mutant phenotype suggests impaired replication progression and increased levels of gross chromosomal rearrangements. Our findings thus indicate a direct role for SUMO in Pol2-mediated DNA synthesis and a molecular basis for Smc5/6-mediated regulation of genome stability.


Assuntos
Proteínas de Ciclo Celular/genética , DNA Polimerase II/genética , DNA/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação/genética , Domínio Catalítico/genética , DNA/genética , Replicação do DNA/genética , Instabilidade Genômica/genética , Lisina/genética , Complexos Multiproteicos/genética , Mutação/genética , Fase S/genética , Saccharomyces cerevisiae/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Telômero/genética
14.
PLoS Genet ; 15(11): e1008427, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31765407

RESUMO

Replication fork stalling and accumulation of single-stranded DNA trigger the S phase checkpoint, a signalling cascade that, in budding yeast, leads to the activation of the Rad53 kinase. Rad53 is essential in maintaining cell viability, but its targets of regulation are still partially unknown. Here we show that Rad53 drives the hyper-SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε, principally following replication forks stalling induced by nucleotide depletion. Pol2 is the main target of SUMOylation within the replisome and its modification requires the SUMO-ligase Mms21, a subunit of the Smc5/6 complex. Moreover, the Smc5/6 complex co-purifies with Pol ε, independently of other replisome components. Finally, we map Pol2 SUMOylation to a single site within the N-terminal catalytic domain and identify a SUMO-interacting motif at the C-terminus of Pol2. These data suggest that the S phase checkpoint regulate Pol ε during replication stress through Pol2 SUMOylation and SUMO-binding ability.


Assuntos
Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , DNA Polimerase II/genética , DNA/biossíntese , Proteína SUMO-1/genética , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação/genética , Domínio Catalítico/genética , DNA/genética , Replicação do DNA/genética , Complexos Multiproteicos/genética , Ligação Proteica , Fase S/genética , Saccharomyces cerevisiae/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética
15.
Crit Rev Biochem Mol Biol ; 54(5): 418-442, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31736364

RESUMO

DNA is constantly exposed to a wide variety of exogenous and endogenous agents, and most DNA lesions inhibit DNA synthesis. To cope with such problems during replication, cells have molecular mechanisms to resume DNA synthesis in the presence of DNA lesions, which are known as DNA damage tolerance (DDT) pathways. The concept of ubiquitination-mediated regulation of DDT pathways in eukaryotes was established via genetic studies in the yeast Saccharomyces cerevisiae, in which two branches of the DDT pathway are regulated via ubiquitination of proliferating cell nuclear antigen (PCNA): translesion DNA synthesis (TLS) and homology-dependent repair (HDR), which are stimulated by mono- and polyubiquitination of PCNA, respectively. Over the subsequent nearly two decades, significant progress has been made in understanding the mechanisms that regulate DDT pathways in other eukaryotes. Importantly, TLS is intrinsically error-prone because of the miscoding nature of most damaged nucleotides and inaccurate replication of undamaged templates by TLS polymerases (pols), whereas HDR is theoretically error-free because the DNA synthesis is thought to be predominantly performed by pol δ, an accurate replicative DNA pol, using the undamaged sister chromatid as its template. Thus, the regulation of the choice between the TLS and HDR pathways is critical to determine the appropriate biological outcomes caused by DNA damage. In this review, we summarize our current understanding of the species-specific regulatory mechanisms of PCNA ubiquitination and how cells choose between TLS and HDR. We then provide a hypothetical model for the spatiotemporal regulation of DDT pathways in human cells.


Assuntos
Dano ao DNA , Reparo do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitinação , Animais , DNA/biossíntese , Humanos , Antígeno Nuclear de Célula em Proliferação/química , Transdução de Sinais , Análise Espaço-Temporal , Ubiquitina/metabolismo
16.
Nucleus ; 10(1): 231-253, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31744372

RESUMO

Constitutive heterochromatin is considered as a functionally inert genome compartment, important for its architecture and stability. How such stable structure is maintained is not well understood. Here, we apply four different visualization schemes to label it and investigate its dynamics during DNA replication and repair. We show that replisomes assemble over the heterochromatin in a temporally ordered manner. Furthermore, heterochromatin undergoes transient decompaction locally at the active sites of DNA synthesis. Using selective laser microirradiation conditions that lead to damage repaired via processive DNA synthesis, we measured similarly local decompaction of heterochromatin. In both cases, we could not observe large-scale movement of heterochromatin to the domain surface. Instead, the processive DNA synthesis machinery assembled at the replication/repair sites. Altogether, our data are compatible with a progression of DNA replication/repair along the chromatin in a dynamic mode with localized and transient decompaction that does not globally remodels the whole heterochromatin compartment.


Assuntos
Reparo do DNA , Replicação do DNA , DNA/biossíntese , Heterocromatina/metabolismo , Animais , Células Cultivadas , DNA/química , Células HeLa , Heterocromatina/química , Humanos , Camundongos
17.
Prog Lipid Res ; 76: 101008, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31626820

RESUMO

N-3 polyunsaturated fatty acids (PUFA) and the numerous families of lipid mediators derived from them collectively regulate numerous biological processes. The mechanisms by which n-3 PUFA regulate biological processes begins with an understanding of the n-3 biosynthetic pathway that starts with alpha-linolenic acid (18:3n-3) and is commonly thought to end with the production of docosahexaenoic acid (DHA, 22:6n-3). However, our understanding of this pathway is not as complete as previously believed. In the current review we provide a background of the evidence supporting the pathway as currently understood and provide updates from recent studies challenging three central dogma of n-3 PUFA metabolism. By building on nearly three decades of research primarily in cell culture and oral dosing studies, recent evidence presented focuses on in vivo kinetic modelling and compound-specific isotope abundance studies in rodents and humans that have been instrumental in expanding our knowledge of the pathway. Specifically, we highlight three main updates to the n-3 PUFA biosynthesis pathway: (1) DHA synthesis rates cannot be as low as previously believed, (2) DHA is both a product and a precursor to tetracosahexaenoic acid (24:6n-3) and (3) increases in EPA in response to DHA supplementation are not the result of increased retroconversion.


Assuntos
DNA/biossíntese , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Graxos Ômega-3/biossíntese , Animais , Humanos
18.
Enzymes ; 45: 139-181, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627876

RESUMO

DNA contains information that must be safeguarded, but also accessed for transcription and replication. To perform replication, eukaryotic cells use the B-family DNA polymerase enzymes Polδ and Polɛ, which are optimized for accuracy, speed, and processivity. The molecular basis of these high-performance characteristics causes these replicative polymerases to fail at sites of DNA damage (lesions), which would lead to genomic instability and cell death. To avoid this, cells possess additional DNA polymerases such as the Y-family of polymerases and the B-family member Polζ that can replicate over sites of DNA damage in a process called translesion synthesis (TLS). While able to replicate over DNA lesions, the TLS polymerases exhibit low-fidelity on undamaged DNA and, consequently, must be prevented from replicating DNA under normal circumstances and recruited only when necessary. The replicative bypass of most types of DNA lesions requires the consecutive action of these specialized TLS polymerases assembled into a dynamic multiprotein complex called the Rev1/Polζ mutasome. To this end, posttranslational modifications and a network of protein-protein interactions mediated by accessory domains/subunits of the TLS polymerases control the assembly and rearrangements of the Rev1/Polζ mutasome and recruitment of TLS proteins to sites of DNA damage. This chapter focuses on the structures and interactions that control these processes underlying the function of the Rev1/Polζ mutasome, as well as the development of small molecule inhibitors of the Rev1/Polζ-dependent TLS holding promise as a potential anticancer therapy.


Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética
19.
Enzymes ; 45: 183-223, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627877

RESUMO

Hexameric DNA helicases involved in the separation of duplex DNA at the replication fork have a universal architecture but have evolved from two separate protein families. The consequences are that the regulation, translocation polarity, strand specificity, and architectural orientation varies between phage/bacteria to that of archaea/eukaryotes. Once assembled and activated for single strand DNA translocation and unwinding, the DNA polymerase couples tightly to the helicase forming a robust replisome complex. However, this helicase-polymerase interaction can be challenged by various forms of endogenous or exogenous agents that can stall the entire replisome or decouple DNA unwinding from synthesis. The consequences of decoupling can be severe, leading to a build-up of ssDNA requiring various pathways for replication fork restart. All told, the hexameric helicase sits prominently at the front of the replisome constantly responding to a variety of obstacles that require transient unwinding/reannealing, traversal of more stable blocks, and alternations in DNA unwinding speed that regulate replisome progression.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , DNA/química , Complexos Multienzimáticos/metabolismo , DNA/metabolismo , DNA de Cadeia Simples/química
20.
Enzymes ; 45: 59-97, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627883

RESUMO

The nucleotide excision repair (NER) system removes a variety of types of helix-distorting lesions from DNA through a dual incision mechanism, in which the damaged nucleotide bases are excised in the form of a small, excised, damage-containing single-stranded DNA oligonucleotide (sedDNA). Damage removal leaves a gap in the DNA template that must then be filled in by the action of a DNA polymerase and ligated to the downstream phosphodiester backbone in the DNA to complete the repair reaction. Defects in damage removal, sedDNA processing, or gap filling have the potential to be mutagenic and lethal to cells, and thus several human pathologies, including cancer and aging, are associated with defects in NER. This review summarizes our current understanding of NER with a focus on the enzymes that excise sedDNAs and restore the duplex DNA to its native state in human cells.


Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA/química , DNA/metabolismo , DNA/biossíntese , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Humanos
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