RESUMO
The Helicase-like Transcription Factor (HLTF) is a member of the SNF2-family of fork remodelers, primarily studied for its capacity to provide DNA Damage Tolerance (DDT) and to induce replication fork reversal (RFR). HLTF is recruited at stalled forks where both its ATPase motor and HIP116 Rad5p N-terminal (HIRAN) domains are necessary for regulating its interaction with DNA. HIRAN bestows specificity to ssDNA 3'-end and imparts branch migration as well as DNA remodeling capabilities facilitating damage repair. Both expression regulation and mutation rate affect HLTF activity. Gene hypermethylation induces loss of HLTF function, in particular in colorectal cancer (CRC), implying a tumour suppressor role. Surprisingly, a correlation between hypermethylation and HLTF mRNA upregulation has also been observed, even within the same cancer type. In many cancers, both complex mutation patterns and the presence of gene Copy Number Variations (CNVs) have been reported. These conditions affect the amount of functional HLTF and question the physiological role of this fork remodeler. This review offers a systematic collection of the presently strewed information regarding HLTF, its structural and functional characteristics, the multiple roles in DDT and the regulation in cancer progression highlighting new research perspectives.
Assuntos
Replicação do DNA , Neoplasias , Fatores de Transcrição , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Reparo do DNA , Dano ao DNA , Animais , Mutação , Variações do Número de Cópias de DNARESUMO
A DNA double-strand break (DSB) is one of the most dangerous types of DNA damage that is repaired largely by homologous recombination or nonhomologous end-joining (NHEJ). The interplay of repair factors at the break directs which pathway is used, and a subset of these factors also function in more mutagenic alternative (alt) repair pathways. Resection is a key event in repair pathway choice and extensive resection, which is a hallmark of homologous recombination, and it is mediated by two nucleases, Exo1 and Dna2. We observed differences in resection and repair outcomes in cells harboring nuclease-dead dna2-1 compared with dna2Δ pif1-m2 that could be attributed to the level of Exo1 recovered at DSBs. Cells harboring dna2-1 showed reduced Exo1 localization, increased NHEJ, and a greater resection defect compared with cells where DNA2 was deleted. Both the resection defect and the increased rate of NHEJ in dna2-1 mutants were reversed upon deletion of KU70 or ectopic expression of Exo1. By contrast, when DNA2 was deleted, Exo1 and Ku70 recovery levels did not change; however, Nej1 increased as did the frequency of alt-end joining/microhomology-mediated end-joining repair. Our findings demonstrate that decreased Exo1 at DSBs contributed to the resection defect in cells expressing inactive Dna2 and highlight the complexity of understanding how functionally redundant factors are regulated in vivo to promote genome stability.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Helicases , Proteínas de Ligação a DNA , Exodesoxirribonucleases , Proteínas de Saccharomyces cerevisiae , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Despite substantial development in medical treatment strategies scientists are struggling to find a cure against spinal cord injury (SCI) which causes long term disability and paralysis. The prime rationale behind it is the enlargement of primary lesion due to an initial trauma to the spinal cord which spreads to the neighbouring spinal tissues It begins from the time of traumatic event happened and extends to hours and even days. It further causes series of biological and functional alterations such as inflammation, excitotoxicity and ischemia, and promotes secondary lesion to the cord which worsens the life of individuals affected by SCI. Oxidative DNA damage is a stern consequence of oxidative stress linked with secondary injury causes oxidative base alterations and strand breaks, which provokes cell death in neurons. It is implausible to stop primary damage however it is credible to halt the secondary lesion and improve the quality of the patient's life to some extent. Therefore it is crucial to understand the hidden perspectives of cell and molecular biology affecting the pathophysiology of SCI. Thus the focus of the review is to connect the missing links and shed light on the oxidative DNA damages and the functional repair mechanisms, as a consequence of the injury in neurons. The review will also probe the significance of neuroprotective strategies in the present scenario. HIGHLIGHTSSpinal cord injury, a pernicious condition, causes excitotoxicity and ischemia, ultimately leading to cell death.Oxidative DNA damage is a consequence of oxidative stress linked with secondary injury, provoking cell death in neurons.Base excision repair (BER) is one of the major repair pathways that plays a crucial role in repairing oxidative DNA damages.Neuroprotective therapies curbing SCI and boosting BER include the usage of pharmacological drugs and other approaches.
Assuntos
Fármacos Neuroprotetores , Traumatismos da Medula Espinal , Humanos , Reparo do DNA , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo , Medula Espinal/patologiaRESUMO
A double -strand break (DSB) is one of the most deleterious forms of DNA damage. In eukaryotic cells, two main repair pathways have evolved to repair DSBs, homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is the predominant pathway of repair in the unicellular eukaryotic organism, S. cerevisiae. However, during replicative aging the relative use of HR and NHEJ shifts in favor of end-joining repair. By monitoring repair events in the HO-DSB system, we find that early in replicative aging there is a decrease in the association of long-range resection factors, Dna2-Sgs1 and Exo1 at the break site and a decrease in DNA resection. Subsequently, as aging progressed, the recovery of Ku70 at DSBs decreased and the break site associated with the nuclear pore complex at the nuclear periphery, which is the location where DSB repair occurs through alternative pathways that are more mutagenic. End-bridging remained intact as HR and NHEJ declined, but eventually it too became disrupted in cells at advanced replicative age. In all, our work provides insight into the molecular changes in DSB repair pathway during replicative aging. HR first declined, resulting in a transient increase in the NHEJ. However, with increased cellular divisions, Ku70 recovery at DSBs and NHEJ subsequently declined. In wild type cells of advanced replicative age, there was a high frequency of repair products with genomic deletions and microhomologies at the break junction, events not observed in young cells which repaired primarily by HR.
Assuntos
Envelhecimento , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae , Envelhecimento/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Helicases/genética , Genômica , Mutação , RecQ Helicases/genética , Reparo de DNA por Recombinação/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
A DNA double strand break (DSB) is primarily repaired by one of two canonical pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ requires no or minimal end processing for ligation, whereas HR requires 5' end resection followed by a search for homology. The main event that determines the mode of repair is the initiation of 5' resection because if resection starts, then NHEJ cannot occur. Nej1 is a canonical NHEJ factor that functions at the cross-roads of repair pathway choice and prior to its function in stimulating Dnl4 ligase. Nej1 competes with Dna2, inhibiting its recruitment to DSBs and thereby inhibiting resection. The highly conserved C-terminal region (CTR) of Nej1 (330-338) is important for two events that drive NHEJ as it stimulates ligation and inhibits resection, but it is dispensable for end-bridging. By combining nej1 point mutants with nuclease-dead dna2-1, we find that Nej1-F335 is essential for end-joining whereas V338 promotes NHEJ indirectly by inhibiting Dna2-mediated resection.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Saccharomyces cerevisiae , DNA/metabolismo , Reparo do DNA por Junção de Extremidades , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The two major pathways of DNA double-strand break repair, nonhomologous end-joining and homologous recombination, are highly conserved from yeast to mammals. The regulation of 5'-DNA resection controls repair pathway choice and influences repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends, and more recently, it was shown to participate in DNA end-bridging and in the inhibition of 5'-resection mediated by the nuclease/helicase complex Dna2-Sgs1. Here, we show that Nej1 interacts with Sae2 to impact DSB repair in three ways. First, we show that Nej1 inhibits interaction of Sae2 with the Mre11-Rad50-Xrs2 complex and Sae2 localization to DSBs. Second, we found that Nej1 inhibits Sae2-dependent recruitment of Dna2 independently of Sgs1. Third, we determined that NEJ1 and SAE2 showed an epistatic relationship for end-bridging, an event that restrains broken DNA ends and reduces the frequency of genomic deletions from developing at the break site. Finally, we demonstrate that deletion of NEJ1 suppressed the synthetic lethality of sae2Δ sgs1Δ mutants, and that triple mutant viability was dependent on Dna2 nuclease activity. Taken together, these findings provide mechanistic insight to how Nej1 functionality inhibits the initiation of DNA resection, a role that is distinct from its involvement in end-joining repair at DSBs.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , DNA/genética , DNA/metabolismo , Reparo do DNA/genética , Endonucleases/genética , Endonucleases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The ribosomal DNA (rDNA) in Saccharomyces cerevisiae is in one tandem repeat array on Chromosome XII. Two regions within each repetitive element, called intergenic spacer 1 (IGS1) and IGS2, are important for organizing the rDNA within the nucleolus. The Smc5/6 complex localizes to IGS1 and IGS2. We show that Smc5/6 has a function in the rDNA beyond its role in homologous recombination (HR) at the replication fork barrier (RFB) located in IGS1. Fob1 is required for optimal binding of Smc5/6 at IGS1 whereas the canonical silencing factor Sir2 is required for its optimal binding at IGS2, independently of Fob1. Through interdependent interactions, Smc5/6 stabilizes Sir2 and Cohibin at both IGS and its recovery at IGS2 is important for nucleolar compaction and transcriptional silencing, which in turn supports rDNA stability and lifespan.
Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA Ribossômico/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , AnimaisRESUMO
RecQ helicases belong to a ubiquitous family of DNA unwinding enzymes that are essential to maintain genome stability by acting at the interface between DNA replication, recombination, and repair. Humans have five different paralogues of RecQ helicases namely RecQ1, BLM, WRN, RecQ4, and RecQ5. Germ-line mutations in these helicases give rise to distinct human genetic disorders, Bloom Syndrome, Werner Syndrome, Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndromes. Other than distinct clinical symptoms, all these genetic disorders show a predisposition to cancer. While the three paralogues BLM, WRN, and RecQ4 are directly associated with syndromes, loss of function of RecQ1 and RecQ5 are also emerging to be causative of various types of cancer. This review summarizes the domain architecture of RecQ helicases and the mutations that are associated with various diseases. Here we observe the occurrence of disease-causing mutations mainly in the catalytic regions of the proteins, and that some of these mutations are common between the respective disorders and cancer. Furthermore, this review discusses the results of several reports that study the role of residues with disease-causing mutations. It also overviews the research focusing on RecQ helicases as potential targets for cancer therapy.
Assuntos
Mutação , RecQ Helicases/genética , Sequência Conservada , Humanos , Neoplasias/enzimologia , Domínios Proteicos , RecQ Helicases/química , RecQ Helicases/metabolismoRESUMO
The DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene- induced carcinogenesis. The complex is integral to all modes of DNA double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S. cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was severely impaired while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Carcinogênese/genética , Saccharomyces cerevisiae/genética , Animais , Dano ao DNA/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Genômica/métodos , Proteína Homóloga a MRE11/genética , Mutação/genética , Células Sf9 , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Non-homologous end joining (NHEJ) and homologous recombination (HR) are the two major pathways of DNA double-strand break (DSB) repair and both are highly conserved from yeast to mammals. Nej1 has a role in DNA end-tethering at a DSB, and the Mre11/Rad50/Xrs2 (MRX) complex is important for its recruitment to the break. Nej1 and Dna2-Sgs1 interact with the C-terminal end of Mre11, which also includes the region where Rad50 binds. By characterizing the functionality of Nej1 in two rad50 mutants, which alter the structural features of MRX, we demonstrate that Nej1 inhibits the binding of Dna2 to Mre11 and Sgs1. Nej1 interactions with Mre11 promote tethering and inhibit hyper-resection, and when these events are compromised, large deletions develop at a DSB. The work indicates that Nej1 provides a layer of regulation to repair pathway choice and is consistent with its role in NHEJ.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA Fúngico/metabolismo , Complexos Multiproteicos/metabolismo , RecQ Helicases/metabolismo , Saccharomyces cerevisiaeRESUMO
Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.
Assuntos
Colorimetria/métodos , DNA/química , Ouro/química , Nanopartículas Metálicas/química , RecQ Helicases/metabolismo , Sequência de Bases , Domínio Catalítico , DNA/genética , Ensaios Enzimáticos/métodos , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Humanos , RecQ Helicases/antagonistas & inibidores , RecQ Helicases/química , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
RecQ helicases are essential in the maintenance of genome stability. Five paralogues (RecQ1, Bloom, Werner, RecQ4, and RecQ5) are found in human cells, with distinct but overlapping roles. Mutations in human RecQ4 give rise to three distinct genetic disorders (Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndromes), characterized by genetic instability, growth deficiency, and predisposition to cancer. Previous studies suggested that RecQ4 was unique because it did not seem to contain a RecQ C-terminal region (RQC) found in the other RecQ paralogues; such a region consists of a zinc domain and a winged helix domain and plays an important role in enzyme activity. However, our recent bioinformatic analysis identified in RecQ4 a putative RQC. To experimentally confirm this hypothesis, we report the purification and characterization of the catalytic core of human RecQ4. Inductively coupled plasma-atomic emission spectrometry detected the unusual presence of two zinc clusters within the zinc domain, consistent with the bioinformatic prediction. Analysis of site-directed mutants, targeting key RQC residues (putative zinc ligands and the aromatic residue predicted to be at the tip of the winged helix ß-hairpin), showed a decrease in DNA binding, unwinding, and annealing, as expected for a functional RQC domain. Low resolution structural information obtained by small angle X-ray scattering data suggests that RecQ4 interacts with DNA in a manner similar to RecQ1, whereas the winged helix domain may assume alternative conformations, as seen in the bacterial enzymes. These combined results experimentally confirm the presence of a functional RQC domain in human RecQ4.
Assuntos
DNA/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , RecQ Helicases/química , RecQ Helicases/metabolismo , Adenosina Trifosfatases/metabolismo , Biologia Computacional , Cristalografia por Raios X , DNA/química , DNA/genética , Bases de Dados de Proteínas , Humanos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Mutação/genética , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , RecQ Helicases/genéticaRESUMO
The RecQ4 helicase belongs to the ubiquitous RecQ family but its exact role in the cell is not completely understood. In addition to the helicase domain, RecQ4 has a unique N-terminal part that is essential for viability and is constituted by a region homologous to the yeast Sld2 replication initiation factor, followed by a cysteine-rich region, predicted to fold as a Zn knuckle. We carried out a structural and biochemical analysis of both the human and Xenopus laevis RecQ4 cysteine-rich regions, and showed by NMR spectroscopy that the Xenopus fragment indeed assumes the canonical Zn knuckle fold, whereas the human sequence remains unstructured, consistent with the mutation of one of the Zn ligands. Both the human and Xenopus Zn knuckles bind to a variety of nucleic acid substrates, with a mild preference for RNA. We also investigated the effect of a segment located upstream the Zn knuckle that is highly conserved and rich in positively charged and aromatic residues, partially overlapping with the C-terminus of the Sld2-like domain. In both the human and Xenopus proteins, the presence of this region strongly enhances binding to nucleic acids. These results reveal novel possible roles of RecQ4 in DNA replication and genome stability.