RESUMO
Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs.
Assuntos
Proteínas de Bactérias/química , Sistemas CRISPR-Cas , Clivagem do DNA , DNA de Cadeia Simples/química , Francisella/química , RNA Guia de Cinetoplastídeos/química , Proteínas de Bactérias/genética , Catálise , DNA de Cadeia Simples/genética , Francisella/genética , Edição de Genes , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Self-catalyzed DNA depurination is a sequence-specific physiological mechanism mediated by spontaneous extrusion of a stem-loop catalytic intermediate. Hydrolysis of the 5'G residue of the 5'GA/TGG loop and of the first 5'A residue of the 5'GAGA loop, together with particular first stem base pairs, specifies their hydrolysis without involving protein, cofactor, or cation. As such, this mechanism is the only known DNA catalytic activity exploited by nature. The consensus sequences for self-depurination of such G- and A-loop residues occur in all genomes examined across the phyla, averaging one site every 2,000-4,000 base pairs. Because apurinic sites are subject to error-prone repair, leading to substitution and short frameshift mutations, they are both a source of genome damage and a means for creating sequence diversity. Their marked overrepresentation in genomes, and largely unchanging density from the lowest to the highest organisms, indicate their selection over the course of evolution. The mutagenicity at such sites in many human genes is associated with loss of function of key proteins responsible for diverse diseases.
Assuntos
Adenina/metabolismo , Síndrome de Bloom/genética , DNA Catalítico/genética , Guanina/metabolismo , Polimorfismo Genético , Síndrome de Werner/genética , Evolução Biológica , Síndrome de Bloom/metabolismo , Síndrome de Bloom/patologia , Catálise , Reparo do DNA , DNA Catalítico/metabolismo , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Humanos , Hidrólise , Sequências Repetidas Invertidas , Mutação , Síndrome de Werner/metabolismo , Síndrome de Werner/patologia , Globinas beta/genética , Globinas beta/metabolismoRESUMO
Telomerase is the essential reverse transcriptase required for linear chromosome maintenance in most eukaryotes. Telomerase supplements the tandem array of simple-sequence repeats at chromosome ends to compensate for the DNA erosion inherent in genome replication. The template for telomerase reverse transcriptase is within the RNA subunit of the ribonucleoprotein complex, which in cells contains additional telomerase holoenzyme proteins that assemble the active ribonucleoprotein and promote its function at telomeres. Telomerase is distinct among polymerases in its reiterative reuse of an internal template. The template is precisely defined, processively copied, and regenerated by release of single-stranded product DNA. New specificities of nucleic acid handling that underlie the catalytic cycle of repeat synthesis derive from both active site specialization and new motif elaborations in protein and RNA subunits. Studies of telomerase provide unique insights into cellular requirements for genome stability, tissue renewal, and tumorigenesis as well as new perspectives on dynamic ribonucleoprotein machines.
Assuntos
Replicação do DNA , DNA de Cadeia Simples/metabolismo , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Telomerase/metabolismo , Telômero/enzimologia , Animais , Domínio Catalítico , DNA de Cadeia Simples/genética , Regulação da Expressão Gênica , Humanos , Repetições de Microssatélites , Conformação de Ácido Nucleico , Oxytricha/genética , Oxytricha/metabolismo , RNA/genética , Ribonucleoproteínas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Telomerase/genética , Telômero/química , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismoRESUMO
Deaminases have important uses in modification detection and genome editing. However, the range of applications is limited by the small number of characterized enzymes. To expand the toolkit of deaminases, we developed an in vitro approach that bypasses a major hurdle with their toxicity in cells. We assayed 175 putative cytosine deaminases on a variety of substrates and found a broad range of activity on double- and single-stranded DNA in various sequence contexts, including CpG-specific deaminases and enzymes without sequence preference. We also characterized enzyme selectivity across six DNA modifications and reported enzymes that do not deaminate modified cytosines. The detailed analysis of diverse deaminases opens new avenues for biotechnological and medical applications. As a demonstration, we developed SEM-seq, a non-destructive single-enzyme methylation sequencing method using a modification-sensitive double-stranded DNA deaminase. The streamlined protocol enables accurate, base-resolution methylome mapping of scarce biological material, including cell-free DNA and 10 pg input DNA.
Assuntos
Citosina Desaminase , Epigenoma , DNA/genética , Citosina , DNA de Cadeia Simples/genética , Citidina Desaminase/genéticaRESUMO
DNA polymerase θ (Polθ) plays a central role in a DNA double-strand break repair pathway termed theta-mediated end joining (TMEJ). TMEJ functions by pairing short-sequence "microhomologies" (MHs) in single-stranded DNA at each end of a break and subsequently initiating DNA synthesis. It is not known how the Polθ helicase domain (HD) and polymerase domain (PD) operate to bring together MHs and facilitate repair. To resolve these transient processes in real time, we utilized in vitro single-molecule FRET approaches and biochemical analyses. We find that the Polθ-HD mediates the initial capture of two ssDNA strands, bringing them in close proximity. The Polθ-PD binds and stabilizes pre-annealed MHs to form a synaptic complex (SC) and initiate repair synthesis. Individual synthesis reactions show that Polθ is inherently non-processive, accounting for complex mutational patterns during TMEJ. Binding of Polθ-PD to stem-loop-forming sequences can substantially limit synapsis, depending on the available dNTPs and sequence context.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Polimerase Dirigida por DNA , DNA Polimerase Dirigida por DNA/metabolismo , Replicação do DNA , DNA de Cadeia Simples/genética , DNA Helicases/genética , Reparo do DNA por Junção de ExtremidadesRESUMO
The metagenome-derived type I-E and type I-F variant CRISPR-associated complex for antiviral defense (Cascade) complexes, fused with HNH domains, precisely cleave target DNA, representing recently identified genome editing tools. However, the underlying working mechanisms remain unknown. Here, structures of type I-FHNH and I-EHNH Cascade complexes at different states are reported. In type I-FHNH Cascade, Cas8fHNH loosely attaches to Cascade head and is adjacent to the 5' end of the target single-stranded DNA (ssDNA). Formation of the full R-loop drives the Cascade head to move outward, allowing Cas8fHNH to detach and rotate â¼150° to accommodate target ssDNA for cleavage. In type I-EHNH Cascade, Cas5eHNH domain is adjacent to the 5' end of the target ssDNA. Full crRNA-target pairing drives the lift of the Cascade head, widening the substrate channel for target ssDNA entrance. Altogether, these analyses into both complexes revealed that crRNA-guided positioning of target DNA and target DNA-induced HNH unlocking are two key factors for their site-specific cleavage of target DNA.
Assuntos
Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Clivagem do DNA , DNA de Cadeia Simples , Edição de Genes , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , Edição de Genes/métodos , Estruturas R-Loop/genética , Microscopia CrioeletrônicaRESUMO
The Bloom syndrome (BLM) helicase is critical for alternative lengthening of telomeres (ALT), a homology-directed repair (HDR)-mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2, commensurate with the appearance of telomere C-strand-specific single-stranded DNA (ssDNA). DNA2 nuclease deficiency increased 5'-flap formation in a BLM-dependent manner, while telomere C-strand, but not G-strand, nicks promoted ALT. These findings define the seminal events in the ALT DNA damage response, linking aberrant telomeric lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers.
Assuntos
Dano ao DNA , Replicação do DNA , RecQ Helicases , Homeostase do Telômero , Telômero , RecQ Helicases/metabolismo , RecQ Helicases/genética , Humanos , Telômero/metabolismo , Telômero/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , Síndrome de Bloom/genética , Síndrome de Bloom/metabolismo , Síndrome de Bloom/enzimologia , Síndrome de Bloom/patologia , Linhagem Celular TumoralRESUMO
Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity.
Assuntos
Bacteriófagos , Microscopia Crioeletrônica , NAD , NAD/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Bacteriófagos/imunologia , Hidrólise , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/imunologia , Sistemas Toxina-Antitoxina/genética , Escherichia coli/virologia , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismoRESUMO
Homology search is a central step of DNA double-strand break (DSB) repair by homologous recombination (HR). How it operates in cells remains elusive. We developed a Hi-C-based methodology to map single-stranded DNA (ssDNA) contacts genome-wide in S. cerevisiae, which revealed two main homology search phases. Initial search conducted by short Rad51-ssDNA nucleoprotein filaments (NPFs) is confined in cis by cohesin-mediated chromatin loop folding. Progressive growth of stiff NPFs enables exploration of distant genomic sites. Long-range resection drives this transition from local to genome-wide search by increasing the probability of assembling extensive NPFs. DSB end-tethering promotes coordinated search by opposite NPFs. Finally, an autonomous genetic element on chromosome III engages the NPF, which stimulates homology search in its vicinity. This work reveals the mechanism of the progressive expansion of homology search that is orchestrated by chromatin organizers, long-range resection, end-tethering, and specialized genetic elements and that exploits the stiff NPF structure conferred by Rad51 oligomerization.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Fúngico , DNA de Cadeia Simples , Rad51 Recombinase , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Cromatina/metabolismo , Cromatina/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , CoesinasRESUMO
Abasic sites are DNA lesions repaired by base excision repair. Cleavage of unrepaired abasic sites in single-stranded DNA (ssDNA) can lead to chromosomal breakage during DNA replication. How rupture of abasic DNA is prevented remains poorly understood. Here, using cryoelectron microscopy (cryo-EM), Xenopus laevis egg extracts, and human cells, we show that RAD51 nucleofilaments specifically recognize and protect abasic sites, which increase RAD51 association rate to DNA. In the absence of BRCA2 or RAD51, abasic sites accumulate as a result of DNA base methylation, oxidation, and deamination, inducing abasic ssDNA gaps that make replicating DNA fibers sensitive to APE1. RAD51 assembled on abasic DNA prevents abasic site cleavage by the MRE11-RAD50 complex, suppressing replication fork breakage triggered by an excess of abasic sites or POLθ polymerase inhibition. Our study highlights the critical role of BRCA2 and RAD51 in safeguarding against unrepaired abasic sites in DNA templates stemming from base alterations, ensuring genomic stability.
Assuntos
Proteína BRCA2 , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples , Rad51 Recombinase , Xenopus laevis , Humanos , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Animais , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Microscopia Crioeletrônica , DNA Polimerase teta , Metilação de DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Proteína Homóloga a MRE11/metabolismo , Proteína Homóloga a MRE11/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genéticaRESUMO
Type IV CRISPR-Cas systems, which are primarily found on plasmids and exhibit a strong plasmid-targeting preference, are the only one of the six known CRISPR-Cas types for which the mechanistic details of their function remain unknown. Here, we provide high-resolution functional snapshots of type IV-A Csf complexes before and after target dsDNA binding, either in the absence or presence of CasDinG, revealing the mechanisms underlying CsfcrRNA complex assembly, "DWN" PAM-dependent dsDNA targeting, R-loop formation, and CasDinG recruitment. Furthermore, we establish that CasDinG, a signature DinG family helicase, harbors ssDNA-stimulated ATPase activity and ATP-dependent 5'-3' DNA helicase activity. In addition, we show that CasDinG unwinds the non-target strand (NTS) and target strand (TS) of target dsDNA from the CsfcrRNA complex. These molecular details advance our mechanistic understanding of type IV-A CRISPR-Csf function and should enable Csf complexes to be harnessed as genome-engineering tools for biotechnological applications.
Assuntos
Proteínas Associadas a CRISPR , DNA , DNA/genética , DNA de Cadeia Simples/genética , Sistemas CRISPR-Cas , Proteínas Associadas a CRISPR/metabolismoRESUMO
Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.
Assuntos
Proteína BRCA2 , Rad51 Recombinase , Humanos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA de Cadeia Simples/genética , DNA/metabolismo , Reparo do DNA , Ligação ProteicaRESUMO
Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases1,2. Most mutations begin as nucleotide mismatches or damage in one of the two strands of the DNA before becoming double-strand mutations if unrepaired or misrepaired3,4. However, current DNA-sequencing technologies cannot accurately resolve these initial single-strand events. Here we develop a single-molecule, long-read sequencing method (Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq)) that achieves single-molecule fidelity for base substitutions when present in either one or both DNA strands. HiDEF-seq also detects cytosine deamination-a common type of DNA damage-with single-molecule fidelity. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes, and derive from them single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumours deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples that are deficient in only polymerase proofreading. We also define a single-strand damage signature for APOBEC3A. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. As double-strand DNA mutations are only the end point of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable studies of how mutations arise in a variety of contexts, especially in cancer and ageing.
Assuntos
Pareamento Incorreto de Bases , Dano ao DNA , DNA de Cadeia Simples , Análise de Sequência de DNA , Imagem Individual de Molécula , Humanos , Envelhecimento/genética , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Pareamento Incorreto de Bases/genética , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Citosina/metabolismo , Desaminação , Dano ao DNA/genética , Reparo de Erro de Pareamento de DNA/genética , Replicação do DNA/genética , DNA de Cadeia Simples/genética , Genoma Mitocondrial/genética , Mutação , Neoplasias/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas , Imagem Individual de Molécula/métodos , Masculino , FemininoRESUMO
Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation1. Here we reveal the activation pathway of the DNA defence module DdmDE system, which rapidly eliminates small, multicopy plasmids from the Vibrio cholerae seventh pandemic strain (7PET)2. Through a combination of cryo-electron microscopy, biochemistry and in vivo plasmid clearance assays, we demonstrate that DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe that the helicase-nuclease DdmD transitions from an autoinhibited, dimeric complex to a monomeric state upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgos utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria.
Assuntos
Proteínas Argonautas , Proteínas de Bactérias , Plasmídeos , Vibrio cholerae , Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Proteínas Argonautas/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Desoxirribonucleases/química , Desoxirribonucleases/metabolismo , Desoxirribonucleases/ultraestrutura , DNA Helicases/química , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Modelos Moleculares , Plasmídeos/genética , Plasmídeos/imunologia , Plasmídeos/metabolismo , Domínios Proteicos , Multimerização Proteica , Vibrio cholerae/genética , Vibrio cholerae/imunologia , Vibrio cholerae/patogenicidadeRESUMO
Although transcription is an essential cellular process, it is paradoxically also a well-recognized cause of genomic instability. R-loops, non-B DNA structures formed when nascent RNA hybridizes to DNA to displace the non-template strand as single-stranded DNA (ssDNA), are partially responsible for this instability. Yet, recent work has begun to elucidate regulatory roles for R-loops in maintaining the genome. In this review, we discuss the cellular contexts in which R-loops contribute to genomic instability, particularly during DNA replication and double-strand break (DSB) repair. We also summarize the evidence that R-loops participate as an intermediate during repair and may influence pathway choice to preserve genomic integrity. Finally, we discuss the immunogenic potential of R-loops and highlight their links to disease should they become pathogenic.
Assuntos
Estruturas R-Loop , Transcrição Gênica , DNA/metabolismo , Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/genética , Instabilidade Genômica , Humanos , Estruturas R-Loop/genéticaRESUMO
RAD51 and the breast cancer suppressor BRCA2 have critical functions in DNA double-strand (dsDNA) break repair by homologous recombination and the protection of newly replicated DNA from nucleolytic degradation. The recombination function of RAD51 requires its binding to single-stranded DNA (ssDNA), whereas binding to dsDNA is inhibitory. Using reconstituted MRE11-, EXO1-, and DNA2-dependent nuclease reactions, we show that the protective function of RAD51 unexpectedly depends on its binding to dsDNA. The BRC4 repeat of BRCA2 abrogates RAD51 binding to dsDNA and accordingly impairs the function of RAD51 in protection. The BRCA2 C-terminal RAD51-binding segment (TR2) acts in a dominant manner to overcome the effect of BRC4. Mechanistically, TR2 stabilizes RAD51 binding to dsDNA, even in the presence of BRC4, promoting DNA protection. Our data suggest that RAD51's dsDNA-binding capacity may have evolved together with its function in replication fork protection and provide a mechanistic basis for the DNA-protection function of BRCA2.
Assuntos
DNA de Cadeia Simples , Rad51 Recombinase , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , DNA/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismoRESUMO
POLQ is a key effector of DSB repair by microhomology-mediated end-joining (MMEJ) and is overexpressed in many cancers. POLQ inhibitors confer synthetic lethality in HR and Shieldin-deficient cancer cells, which has been proposed to reflect a critical dependence on the DSB repair pathway by MMEJ. Whether POLQ also operates independent of MMEJ remains unexplored. Here, we show that POLQ-deficient cells accumulate post-replicative ssDNA gaps upon BRCA1/2 loss or PARP inhibitor treatment. Biochemically, cooperation between POLQ helicase and polymerase activities promotes RPA displacement and ssDNA-gap fill-in, respectively. POLQ is also capable of microhomology-mediated gap skipping (MMGS), which generates deletions during gap repair that resemble the genomic scars prevalent in POLQ overexpressing cancers. Our findings implicate POLQ in mutagenic post-replicative gap sealing, which could drive genome evolution in cancer and whose loss places a critical dependency on HR for gap protection and repair and cellular viability.
Assuntos
Quebras de DNA de Cadeia Dupla , Neoplasias , Humanos , Replicação do DNA/genética , Instabilidade Genômica , DNA de Cadeia Simples/genética , Mutações Sintéticas Letais , Reparo do DNA por Junção de Extremidades , Neoplasias/genéticaRESUMO
Retrotransposons are highly enriched in the animal genome1-3. The activation of retrotransposons can rewrite host DNA information and fundamentally impact host biology1-3. Although developmental activation of retrotransposons can offer benefits for the host, such as against virus infection, uncontrolled activation promotes disease or potentially drives ageing1-5. After activation, retrotransposons use their mRNA as templates to synthesize double-stranded DNA for making new insertions in the host genome1-3,6. Although the reverse transcriptase that they encode can synthesize the first-strand DNA1-3,6, how the second-strand DNA is generated remains largely unclear. Here we report that retrotransposons hijack the alternative end-joining (alt-EJ) DNA repair process of the host for a circularization step to synthesize their second-strand DNA. We used Nanopore sequencing to examine the fates of replicated retrotransposon DNA, and found that 10% of them achieve new insertions, whereas 90% exist as extrachromosomal circular DNA (eccDNA). Using eccDNA production as a readout, further genetic screens identified factors from alt-EJ as essential for retrotransposon replication. alt-EJ drives the second-strand synthesis of the long terminal repeat retrotransposon DNA through a circularization process and is therefore necessary for eccDNA production and new insertions. Together, our study reveals that alt-EJ is essential in driving the propagation of parasitic genomic retroelements. Our study uncovers a conserved function of this understudied DNA repair process, and provides a new perspective to understand-and potentially control-the retrotransposon life cycle.
Assuntos
Reparo do DNA por Junção de Extremidades , Replicação do DNA , DNA Circular , Parasitos , Retroelementos , Animais , Retroelementos/genética , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Moldes Genéticos , DNA Circular/biossíntese , DNA Circular/genética , DNA Circular/metabolismo , DNA de Cadeia Simples/biossíntese , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Parasitos/genética , Genoma/genéticaRESUMO
Homologous recombination (HR) fulfils a pivotal role in the repair of DNA double-strand breaks and collapsed replication forks1. HR depends on the products of several paralogues of RAD51, including the tetrameric complex of RAD51B, RAD51C, RAD51D and XRCC2 (BCDX2)2. BCDX2 functions as a mediator of nucleoprotein filament assembly by RAD51 and single-stranded DNA (ssDNA) during HR, but its mechanism remains undefined. Here we report cryogenic electron microscopy reconstructions of human BCDX2 in apo and ssDNA-bound states. The structures reveal how the amino-terminal domains of RAD51B, RAD51C and RAD51D participate in inter-subunit interactions that underpin complex formation and ssDNA-binding specificity. Single-molecule DNA curtain analysis yields insights into how BCDX2 enhances RAD51-ssDNA nucleoprotein filament assembly. Moreover, our cryogenic electron microscopy and functional analyses explain how RAD51C alterations found in patients with cancer3-6 inactivate DNA binding and the HR mediator activity of BCDX2. Our findings shed light on the role of BCDX2 in HR and provide a foundation for understanding how pathogenic alterations in BCDX2 impact genome repair.
Assuntos
Proteínas de Ligação a DNA , Recombinação Homóloga , Complexos Multiproteicos , Humanos , Microscopia Crioeletrônica , Replicação do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Neoplasias/genética , Nucleoproteínas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Rad51 Recombinase/ultraestrutura , Especificidade por SubstratoRESUMO
Accurate DNA replication is constantly threatened by DNA lesions arising from endogenous and exogenous sources. Specialized DNA replication stress response pathways ensure replication fork progression in the presence of DNA lesions with minimal delay in fork elongation. These pathways broadly include translesion DNA synthesis, template switching, and replication fork repriming. Here, we discuss recent advances toward our understanding of the mechanisms that regulate the fine-tuned balance between these different replication stress response pathways. We also discuss the molecular pathways required to fill single-stranded DNA gaps that accumulate throughout the genome after repriming and the biological consequences of using repriming instead of other DNA damage tolerance pathways on genome integrity and cell fitness.