RESUMO
DNA polymerase θ (Pol θ) is a DNA repair enzyme widely conserved in animals and plants. Pol θ uses short DNA sequence homologies to initiate repair of double-strand breaks by theta-mediated end joining. The DNA polymerase domain of Pol θ is at the C terminus and is connected to an N-terminal DNA helicase-like domain by a central linker. Pol θ is crucial for maintenance of damaged genomes during development, protects DNA against extensive deletions, and limits loss of heterozygosity. The cost of using Pol θ for genome protection is that a few nucleotides are usually deleted or added at the repair site. Inactivation of Pol θ often enhances the sensitivity of cells to DNA strand-breaking chemicals and radiation. Since some homologous recombination-defective cancers depend on Pol θ for growth, inhibitors of Pol θ may be useful in treating such tumors.
Assuntos
DNA Polimerase Dirigida por DNA , Neoplasias , Animais , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Reparo do DNA por Junção de Extremidades/genética , DNA , Dano ao DNA/genética , Neoplasias/genética , DNA Polimerase tetaRESUMO
Nonscheduled R loops represent a major source of DNA damage and replication stress. Cells have different ways to prevent R-loop accumulation. One mechanism relies on the conserved THO complex in association with cotranscriptional RNA processing factors including the RNA-dependent ATPase UAP56/DDX39B and histone modifiers such as the SIN3 deacetylase in humans. We investigated the function of UAP56/DDX39B in R-loop removal. We show that UAP56 depletion causes R-loop accumulation, R-loop-mediated genome instability, and replication fork stalling. We demonstrate an RNA-DNA helicase activity in UAP56 and show that its overexpression suppresses R loops and genome instability induced by depleting five different unrelated factors. UAP56/DDX39B localizes to active chromatin and prevents the accumulation of RNA-DNA hybrids over the entire genome. We propose that, in addition to its RNA processing role, UAP56/DDX39B is a key helicase required to eliminate harmful cotranscriptional RNA structures that otherwise would block transcription and replication.
Assuntos
RNA Helicases DEAD-box/metabolismo , Genoma/genética , Estruturas R-Loop/genética , Transcrição Gênica/genética , Cromatina/metabolismo , RNA Helicases DEAD-box/genética , Expressão Gênica/genética , Instabilidade Genômica/genética , Humanos , Células K562RESUMO
R loops arise from hybridization of RNA transcripts with template DNA during transcription. Unrepaired R loops lead to transcription-replication collisions, causing DNA damage and genomic instability. In this issue of Genes & Development, Pérez-Calero and colleagues (pp. 898-912) identify UAP56 as a cotranscriptional RNA-DNA helicase that unwinds R loops. They found that UAP56 helicase activity is required to remove R loops formed from different sources and prevent R-loop accumulation genome-wide at actively transcribed genes.
Assuntos
Genoma/genética , Estruturas R-Loop/genética , Transcrição Gênica/genética , Cromatina/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Instabilidade Genômica/genética , Humanos , Células K562RESUMO
Several microbial genomes lack textbook-defined essential genes. If an essential gene is absent from a genome, then an evolutionarily independent gene of unknown function complements its function. Here, we identified frequent nonhomologous replacement of an essential component of DNA replication initiation, a replicative helicase loader gene, in Vibrionaceae. Our analysis of Vibrionaceae genomes revealed two genes with unknown function, named vdhL1 and vdhL2, that were substantially enriched in genomes without the known helicase-loader genes. These genes showed no sequence similarities to genes with known function but encoded proteins structurally similar with a viral helicase loader. Analyses of genomic syntenies and coevolution with helicase genes suggested that vdhL1/2 encodes a helicase loader. The in vitro assay showed that Vibrio harveyi VdhL1 and Vibrio ezurae VdhL2 promote the helicase activity of DnaB. Furthermore, molecular phylogenetics suggested that vdhL1/2 were derived from phages and replaced an intrinsic helicase loader gene of Vibrionaceae over 20 times. This high replacement frequency implies the host's advantage in acquiring a viral helicase loader gene.
Assuntos
DNA Helicases , Replicação do DNA , Filogenia , Vibrionaceae , Vibrionaceae/genética , Vibrionaceae/enzimologia , DNA Helicases/metabolismo , DNA Helicases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Bacteriófagos/genética , Bacteriófagos/enzimologia , Evolução Molecular , Genoma Bacteriano , DnaB Helicases/metabolismo , DnaB Helicases/genética , Vibrio/genética , Vibrio/enzimologiaRESUMO
DNA replication in Escherichia coli starts with loading of the replicative helicase, DnaB, onto DNA. This reaction requires the DnaC loader protein, which forms a 6:6 complex with DnaB and opens a channel in the DnaB hexamer through which single-stranded DNA is thought to pass. During replication, replisomes frequently encounter DNA damage and nucleoprotein complexes that can lead to replication fork collapse. Such events require DnaB re-loading onto DNA to allow replication to continue. Replication restart proteins mediate this process by recruiting DnaB6/DnaC6 to abandoned DNA replication forks. Several dnaC mutations that bypass the requirement for replication restart proteins or that block replication restart have been identified in E. coli. To better understand how these DnaC variants function, we have purified and characterized the protein products of several such alleles. Unlike wild-type DnaC, three of the variants (DnaC 809, DnaC 809,820, and DnaC 811) can load DnaB onto replication forks bound by single-stranded DNA-binding protein. DnaC 809 can also load DnaB onto double-stranded DNA. These results suggest that structural changes in the variant DnaB6/DnaC6 complexes expand the range of DNA substrates that can be used for DnaB loading, obviating the need for the existing replication restart pathways. The protein product of dnaC1331, which phenocopies deletion of the priB replication restart gene, blocks loading through the major restart pathway in vitro. Overall, the results of our study highlight the utility of bacterial DnaC variants as tools for probing the regulatory mechanisms that govern replicative helicase loading.
Assuntos
Replicação do DNA , DnaB Helicases , Proteínas de Escherichia coli , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Escherichia coli/genética , DnaB Helicases/metabolismo , DnaB Helicases/genética , DnaB Helicases/química , DNA Bacteriano/metabolismo , DNA Bacteriano/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , MutaçãoRESUMO
Mycobacterium tuberculosis (Mtb) causes tuberculosis and, during infection, is exposed to reactive oxygen species and reactive nitrogen intermediates from the host immune response that can cause DNA damage. UvrD-like proteins are involved in DNA repair and replication and belong to the SF1 family of DNA helicases that use ATP hydrolysis to catalyze DNA unwinding. In Mtb, there are two UvrD-like enzymes, where UvrD1 is most closely related to other family members. Previous studies have suggested that UvrD1 is exclusively monomeric; however, it is well known that Escherichia coli UvrD and other UvrD family members exhibit monomer-dimer equilibria and unwind as dimers in the absence of accessory factors. Here, we reconcile these incongruent studies by showing that Mtb UvrD1 exists in monomer, dimer, and higher-order oligomeric forms, where dimerization is regulated by redox potential. We identify a 2B domain cysteine, conserved in many Actinobacteria, that underlies this effect. We also show that UvrD1 DNA-unwinding activity correlates specifically with the dimer population and is thus titrated directly via increasing positive (i.e., oxidative) redox potential. Consistent with the regulatory role of the 2B domain and the dimerization-based activation of DNA unwinding in UvrD family helicases, these results suggest that UvrD1 is activated under oxidizing conditions when it may be needed to respond to DNA damage during infection.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Helicases/metabolismo , Reparo do DNA/fisiologia , Mycobacterium tuberculosis/genética , Proteínas de Bactérias/genética , Cisteína/química , DNA/genética , DNA/metabolismo , Dano ao DNA , DNA Helicases/genética , Reparo do DNA/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples , Dimerização , Oxirredução , Ligação Proteica , Domínios Proteicos/genéticaRESUMO
DNA2 nuclease-helicase functions in DNA replication and recombination. This requires the nuclease of DNA2, while, in contrast, the role of the helicase activity has been unclear. We now show that the motor activity of both recombinant yeast and human DNA2 promotes efficient degradation of long stretches of ssDNA, particularly in the presence of the replication protein A. This degradation is further stimulated by a direct interaction with a cognate RecQ family helicase, which functions with DNA2 in DNA end resection to initiate homologous recombination. Consequently, helicase-deficient yeast dna2 K1080E cells display reduced resection speed of HO-induced DNA double-strand breaks. These results support a model of DNA2 and the RecQ family helicase partner forming a bidirectional motor machine, where the RecQ family helicase is the lead helicase, and the motor of DNA2 functions as a ssDNA translocase to promote degradation of 5'-terminated DNA.
Assuntos
Reparo do DNA por Junção de Extremidades/fisiologia , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Reparo do DNA por Junção de Extremidades/genética , Recombinação Homóloga , Humanos , RecQ Helicases/metabolismo , Proteínas Recombinantes/genética , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
Escherichia coli YoaA aids in the resolution of DNA damage that halts DNA synthesis in vivo in conjunction with χ, an accessory subunit of DNA polymerase III. YoaA and χ form a discrete complex separate from the DNA polymerase III holoenzyme, but little is known about how YoaA and χ work together to help the replication fork overcome damage. Although YoaA is predicted to be an iron-sulfur helicase in the XPD/Rad3 helicase family based on sequence analysis, the biochemical activities of YoaA have not been described. Here, we characterize YoaA and show that purified YoaA contains iron. YoaA and χ form a complex that is stable through three chromatographic steps, including gel filtration chromatography. When overexpressed in the absence of χ, YoaA is mostly insoluble. In addition, we show the YoaA-χ complex has DNA-dependent ATPase activity. Our measurement of the YoaA-χ helicase activity illustrates for the first time YoaA-χ translocates on ssDNA in the 5' to 3' direction and requires a 5' single-stranded overhang, or ssDNA gap, for DNA/DNA unwinding. Furthermore, YoaA-χ preferentially unwinds forked duplex DNA that contains both 3' and 5' single-stranded overhangs versus duplex DNA with only a 5' overhang. Finally, we demonstrate YoaA-χ can unwind damaged DNA that contains an abasic site or damage on 3' ends that stall replication extension. These results are the first biochemical evidence demonstrating YoaA is a bona fide iron-sulfur helicase, and we further propose the physiologically relevant form of the helicase is YoaA-χ.
Assuntos
DNA Helicases , DNA Polimerase III , Proteínas de Escherichia coli , Escherichia coli , DNA Helicases/metabolismo , DNA Polimerase III/genética , Replicação do DNA , DNA de Cadeia Simples , Escherichia coli/metabolismo , Ferro , Proteínas de Escherichia coli/metabolismo , Reparo do DNARESUMO
The SLFN11 gene participates in cell fate decision following cancer chemotherapy and encodes the N-terminal ribonuclease (RNase) domain and the C-terminal helicase/ATPase domain. How these domains contribute to the chemotherapeutic response remains controversial. Here, we expressed SLFN11 containing mutations in two critical residues required for RNase activity in SLFN11-/- cells. We found that this mutant was still able to suppress DNA damage tolerance, destabilized the stalled replication forks, and perturbed recruitment of the fork protector RAD51. In contrast, we confirmed that the helicase domain was essential to accelerate fork degradation. The fork degradation by the RNase mutant was dependent on both DNA2 and MRE11 nuclease, but not on MRE11's novel interactor FXR1. Collectively, these results supported the view that the RNase domain function is dispensable for SLFN11 to mediate cell fate decision during replication stress response.
Assuntos
Replicação do DNA , Ribonucleases , Ribonucleases/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Mutação , Dano ao DNARESUMO
The guanine-rich stretch of single-stranded DNA (ssDNA) forms a G-quadruplex (G4) in a fraction of genic and intergenic chromosomal regions. The probability of G4 formation increases during events causing ssDNA generation, such as transcription and replication. In turn, G4 abrogates these events, leading to DNA damage. DHX36 unwinds G4-DNA in vitro and in human cells. However, its spatial correlation with G4-DNA in vivo and its role in genome maintenance remain unclear. Here, we demonstrate a connection between DHX36 and G4-DNA and its implications for genomic integrity. The nuclear localization of DHX36 overlapped with that of G4-DNA, RNA polymerase II, and a splicing-related factor. Depletion of DHX36 resulted in accumulated DNA damage, slower cell growth, and enhanced cell growth inhibition upon treatment with a G4-stabilizing compound; DHX36 expression reversed these defects. In contrast, the reversal upon expression of DHX36 mutants that could not bind G4 was imperfect. Thus, DHX36 may suppress DNA damage by promoting the clearance of G4-DNA for cell growth and survival. Our findings deepen the understanding of G4 resolution in the maintenance of genomic integrity.
RESUMO
The T7 primase-helicase plays a pivotal role in the replication of T7 DNA. Using affinity isolation of peptide-nucleic acid crosslinks and mass spectrometry, we identify protein regions in the primase-helicase and T7 DNA polymerase that form contacts with the RNA primer and DNA template. The contacts between nucleic acids and the primase domain of the primase-helicase are centered in the RNA polymerase subdomain of the primase domain, in a cleft between the N-terminal subdomain and the topoisomerase-primase fold. We demonstrate that residues along a beta sheet in the N-terminal subdomain that contacts the RNA primer are essential for phage growth and primase activity in vitro. Surprisingly, we found mutations in the primase domain that had a dramatic effect on the helicase. Substitution of a residue conserved in other DnaG-like enzymes, R84A, abrogates both primase and helicase enzymatic activities of the T7 primase-helicase. Alterations in this residue also decrease binding of the primase-helicase to ssDNA. However, mass photometry measurements show that these mutations do not interfere with the ability of the protein to form the active hexamer.
Assuntos
Bacteriófago T7 , DNA Helicases , DNA Primase , DNA , Proteínas Virais , Sequência de Aminoácidos , Bacteriófago T7/enzimologia , DNA/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , DNA Primase/química , DNA Primase/genética , DNA Primase/metabolismo , Mutação , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
DNA replication is highly regulated and primarily controlled at the step of initiation. In bacteria, the replication initiator DnaA and the origin of replication oriC are the primary targets of regulation. Perturbations that increase or decrease replication initiation can cause a decrease in cell fitness. We found that multiple mechanisms, including an increase in replication elongation and a decrease in replication initiation, can compensate for lethal over-initiation. We found that in Bacillus subtilis, under conditions of rapid growth, loss of yabA, a negative regulator of replication initiation, caused a synthetic lethal phenotype when combined with the dnaA1 mutation that also causes replication over-initiation. We isolated several classes of suppressors that restored viability to dnaA1 ∆yabA double mutants. Some suppressors (relA, nrdR) stimulated replication elongation. Others (dnaC, cshA) caused a decrease in replication initiation. One class of suppressors decreased replication initiation in the dnaA1 ∆yabA mutant by causing a decrease in the amount of the replicative helicase, DnaC. We found that decreased levels of helicase in otherwise wild-type cells were sufficient to decrease replication initiation during rapid growth, indicating that the replicative helicase is limiting for replication initiation. Our results highlight the multiple mechanisms cells use to regulate DNA replication.
Assuntos
Proteínas de Bactérias , Proteínas de Ligação a DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Bactérias/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Replicação do DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Origem de ReplicaçãoRESUMO
There are multiple assays available that can provide insight into the biochemical mechanism of DNA helicases. For the first 22 years since their discovery, bulk-phase assays were used. These include gel-based, spectrophotometric, and spectrofluorometric assays that revealed many facets of these enzymes. From 2001, single-molecule studies have contributed additional insight into these DNA nanomachines to reveal details on energy coupling, step size, processivity as well as unique aspects of individual enzyme behavior that were masked in the averaging inherent in ensemble studies. In this review, important aspects of the study of helicases are discussed including beginning with active, nuclease-free enzyme, followed by several bulk-phase approaches that have been developed and still find widespread use today. Finally, two single-molecule approaches are discussed, and the resulting findings are related to the results obtained in bulk-phase studies.
Assuntos
DNA Helicases , DNA , DNA/química , DNA Helicases/química , DNA Helicases/genéticaRESUMO
DNA helicases function in many types of nucleic acid transactions, and as such, they are vital for genome integrity. Although they are often considered individually, work from many groups demonstrates that these enzymes often genetically and biochemically interact in vivo. Here, we highlight methods to interrogate such interactions among the PIF1 (Pif1 and Rrm3) and RecQ (Hrq1 and Sgs1) family helicases in Saccharomyces cerevisiae. The interactions among these enzymes were investigated in vivo using deletion and inactivation alleles with a gross-chromosomal rearrangement (GCR) assay. Further, wild-type and inactive recombinant proteins were used to determine the effects of the helicases on telomerase activity in vitro. We found that synergistic increases in GCR rates often occur in double vs. single mutants, suggesting that the helicases function in distinct genome integrity pathways. Further, the recombinant helicases can function together in vitro to modulate telomerase activity. Overall, the data suggest that the interactions among the members of these DNA helicase families are multipartite and argue for a comprehensive systems biology approach to fully elucidate the physiological interplay between these enzymes.
Assuntos
DNA Helicases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Telomerase , DNA Helicases/genética , DNA Helicases/metabolismo , RecQ Helicases/genética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Telomerase/metabolismoRESUMO
DNA helicase RECQ1 (also known as RECQL or RECQL1) is a candidate breast cancer susceptibility gene significantly correlated with clinical outcomes of sporadic breast cancer patients. Prior studies have suggested that RECQ1 maintains genomic stability by regulating a wide variety of core cellular functions including DNA replication, DNA damage response, and transcription. However, it is unclear which, if any, of these are the primary functions of RECQ1 as related to its role in suppressing breast cancer. We describe here an unbiased integrative genomics approach that enabled us to discover a previously unknown regulatory role of RECQ1 in promoting Estrogen Receptor alpha (ERα) expression and the expression of specific ERα target genes in ER positive breast cancer cells. We discuss potential future applications of similar experimental strategies in advancing the mechanistic understanding and elucidating specific new details of genome-wide functions of RECQ1 and other RecQ helicases in maintaining genomic stability and preventing cancer.
Assuntos
Neoplasias da Mama , RecQ Helicases , Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Feminino , Predisposição Genética para Doença , Instabilidade Genômica , Humanos , RecQ Helicases/genéticaRESUMO
Mammalian mitochondria contain multiple copies of a circular, double-stranded DNA genome (mtDNA) that codes for subunits of the oxidative phosphorylation machinery. Mutations in mtDNA cause a number of rare, human disorders and are also associated with more common conditions, such as neurodegeneration and biological aging. In this review, we discuss our current understanding of mtDNA replication in mammalian cells and how this process is regulated. We also discuss how deletions can be formed during mtDNA replication.
Assuntos
DNA Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA/genética , Replicação do DNA/fisiologia , DNA Mitocondrial/genética , Humanos , Proteínas Mitocondriais/genéticaRESUMO
Natural transformation is one of the major mechanisms of horizontal gene transfer in bacterial populations and has been demonstrated in numerous species of bacteria. Despite the prevalence of natural transformation, much of the molecular mechanism remains unexplored. One major outstanding question is how the cell powers DNA import, which is rapid and highly processive. ComFA is one of a few proteins required for natural transformation in Gram-positive bacteria. Its structural resemblance to the DEAD box helicase family has led to a long-held hypothesis that ComFA acts as a motor to help drive DNA import into the cytosol. Here, we explored the helicase and translocase activity of ComFA to address this hypothesis. We followed the DNA-dependent ATPase activity of ComFA and, combined with mathematical modeling, demonstrated that ComFA likely translocates on single-stranded DNA from 5' to 3'. However, this translocase activity does not lead to DNA unwinding under the conditions we tested. Further, we analyzed the ATPase cycle of ComFA and found that ATP hydrolysis stimulates the release of DNA, providing a potential mechanism for translocation. These findings help define the molecular contribution of ComFA to natural transformation and support the conclusion that ComFA plays a key role in powering DNA uptake. IMPORTANCE Competence, or the ability of bacteria to take up and incorporate foreign DNA in a process called natural transformation, is common in the bacterial kingdom. Research in several bacterial species suggests that long, contiguous stretches of DNA are imported into cells in a processive manner, but how bacteria power transformation remains unclear. Our finding that ComFA, a DEAD box helicase required for competence in Gram-positive bacteria, translocates on single-stranded DNA from 5' to 3', supports the long-held hypothesis that ComFA may be the motor powering DNA transport during natural transformation. Moreover, ComFA may be a previously unidentified type of DEAD box helicase-one with the capability of extended translocation on single-stranded DNA.
Assuntos
Adenosina Trifosfatases , DNA de Cadeia Simples , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , RNA Helicases DEAD-box/metabolismo , DNA , DNA Helicases/metabolismo , DNA de Cadeia Simples/genéticaRESUMO
A fundamental requirement for life is the replication of an organism's DNA. Studies in Escherichia coli and Bacillus subtilis have set the paradigm for DNA replication in bacteria. During replication initiation in E. coli and B. subtilis, the replicative helicase is loaded onto the DNA at the origin of replication by an ATPase helicase loader. However, most bacteria do not encode homologs to the helicase loaders in E. coli and B. subtilis. Recent work has identified the DciA protein as a predicted helicase operator that may perform a function analogous to the helicase loaders in E. coli and B. subtilis. DciA proteins, which are defined by the presence of a DUF721 domain (termed the DciA domain herein), are conserved in most bacteria but have only been studied in mycobacteria and gammaproteobacteria (Pseudomonas aeruginosa and Vibrio cholerae). Sequences outside the DciA domain in Mycobacterium tuberculosis DciA are essential for protein function but are not conserved in the P. aeruginosa and V. cholerae homologs, raising questions regarding the conservation and evolution of DciA proteins across bacterial phyla. To comprehensively define the DciA protein family, we took a computational evolutionary approach and analyzed the domain architectures and sequence properties of DciA domain-containing proteins across the tree of life. These analyses identified lineage-specific domain architectures among DciA homologs, as well as broadly conserved sequence-structural motifs. The diversity of DciA proteins represents the evolution of helicase operation in bacterial DNA replication and highlights the need for phylum-specific analyses of this fundamental biological process. IMPORTANCE Despite the fundamental importance of DNA replication for life, this process remains understudied in bacteria outside Escherichia coli and Bacillus subtilis. In particular, most bacteria do not encode the helicase-loading proteins that are essential in E. coli and B. subtilis for DNA replication. Instead, most bacteria encode a DciA homolog that likely constitutes the predominant mechanism of helicase operation in bacteria. However, it is still unknown how DciA structure and function compare across diverse phyla that encode DciA proteins. In this study, we performed computational evolutionary analyses to uncover tremendous diversity among DciA homologs. These studies provide a significant advance in our understanding of an essential component of the bacterial DNA replication machinery.
Assuntos
Proteínas de Bactérias , Escherichia coli , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , DNA/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
The complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. In Escherichia coli, replication termination, the final stage of the duplication process, is confined to the "replication fork trap" region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomic ter sites. Termination typically occurs away from Tus-ter complexes, but they become part of the fork fusion process when a delay to one replisome allows the second replisome to travel more than halfway around the chromosome. In this instance, replisome progression is blocked at the nonpermissive interface of the Tus-ter complex, termination then occurs when a converging replisome meets the permissive interface. To investigate the consequences of replication fork fusion at Tus-ter complexes, we established a plasmid-based replication system where we could mimic the termination process at Tus-ter complexes in vitro. We developed a termination mapping assay to measure leading strand replication fork progression and demonstrate that the DNA template is under-replicated by 15 to 24 bases when replication forks fuse at Tus-ter complexes. This gap could not be closed by the addition of lagging strand processing enzymes or by the inclusion of several helicases that promote DNA replication. Our results indicate that accurate fork fusion at Tus-ter barriers requires further enzymatic processing, highlighting large gaps that still exist in our understanding of the final stages of chromosome duplication and the evolutionary advantage of having a replication fork trap.
Assuntos
Replicação do DNA , DNA Bacteriano , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Escherichia coli , DNA Bacteriano/biossíntese , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
GGGGCC (G4C2) hexanucleotide repeat expansions in the endosomal trafficking gene C9orf72 are the most common genetic cause of ALS and frontotemporal dementia. Repeat-associated non-AUG (RAN) translation of this expansion through near-cognate initiation codon usage and internal ribosomal entry generates toxic proteins that accumulate in patients' brains and contribute to disease pathogenesis. The helicase protein DEAH-box helicase 36 (DHX36-G4R1) plays active roles in RNA and DNA G-quadruplex (G4) resolution in cells. As G4C2 repeats are known to form G4 structures in vitro, we sought to determine the impact of manipulating DHX36 expression on repeat transcription and RAN translation. Using a series of luciferase reporter assays both in cells and in vitro, we found that DHX36 depletion suppresses RAN translation in a repeat length-dependent manner, whereas overexpression of DHX36 enhances RAN translation from G4C2 reporter RNAs. Moreover, upregulation of RAN translation that is typically triggered by integrated stress response activation is prevented by loss of DHX36. These results suggest that DHX36 is active in regulating G4C2 repeat translation, providing potential implications for therapeutic development in nucleotide repeat expansion disorders.