RESUMEN
Homologous recombination (HR) helps maintain genome integrity, and HR defects give rise to disease, especially cancer. During HR, damaged DNA must be aligned with an undamaged template through a process referred to as the homology search. Despite decades of study, key aspects of this search remain undefined. Here, we use single-molecule imaging to demonstrate that Rad54, a conserved Snf2-like protein found in all eukaryotes, switches the search from the diffusion-based pathways characteristic of the basal HR machinery to an active process in which DNA sequences are aligned via an ATP-dependent molecular motor-driven mechanism. We further demonstrate that Rad54 disrupts the donor template strands, enabling the search to take place within a migrating DNA bubble-like structure that is bound by replication protein A (RPA). Our results reveal that Rad54, working together with RPA, fundamentally alters how DNA sequences are aligned during HR.
Asunto(s)
Adenosina Trifosfato/genética , ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , ADN/genética , Recombinación Homóloga/genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/genética , Daño del ADN/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Hidrólisis , Saccharomyces cerevisiae/genética , Alineación de Secuencia/métodosRESUMEN
Mutations in the BRCA1 and BRCA2 genes predispose afflicted individuals to breast, ovarian, and other cancers. The BRCA-encoded products form complexes with other tumor suppressor proteins and with the recombinase enzyme RAD51 to mediate chromosome damage repair by homologous recombination and also to protect stressed DNA replication forks against spurious nucleolytic attrition. Understanding how the BRCA tumor suppressor network executes its biological functions would provide the foundation for developing targeted cancer therapeutics, but progress in this area has been greatly hampered by the challenge of obtaining purified BRCA complexes for mechanistic studies. In this article, we review how recent effort begins to overcome this technical challenge, leading to functional and structural insights into the biochemical attributes of these complexes and the multifaceted roles that they fulfill in genome maintenance. We also highlight the major mechanistic questions that remain.
Asunto(s)
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias de la Mama/genética , Redes Reguladoras de Genes , Recombinasa Rad51/genética , Reparación del ADN por Recombinación , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Proteína BRCA1/química , Proteína BRCA1/metabolismo , Proteína BRCA2/química , Proteína BRCA2/metabolismo , Sitios de Unión , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , ADN/química , ADN/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , Replicación del ADN , Femenino , Genoma Humano , Inestabilidad Genómica , Humanos , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
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.
Asunto(s)
Daño del ADN , Replicación del ADN , RecQ Helicasas , Homeostasis del Telómero , Telómero , RecQ Helicasas/metabolismo , RecQ Helicasas/genética , Humanos , Telómero/metabolismo , Telómero/genética , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/genética , Proteína Nuclear Ligada al Cromosoma X/genética , Proteína Nuclear Ligada al Cromosoma X/metabolismo , ADN Helicasas/metabolismo , ADN Helicasas/genética , Síndrome de Bloom/genética , Síndrome de Bloom/metabolismo , Síndrome de Bloom/enzimología , Síndrome de Bloom/patología , Línea Celular TumoralRESUMEN
The tumor-suppressor breast cancer 1 (BRCA1) in complex with BRCA1-associated really interesting new gene (RING) domain 1 (BARD1) is a RING-type ubiquitin E3 ligase that modifies nucleosomal histone and other substrates. The importance of BRCA1-BARD1 E3 activity in tumor suppression remains highly controversial, mainly stemming from studying mutant ligase-deficient BRCA1-BARD1 species that we show here still retain significant ligase activity. Using full-length BRCA1-BARD1, we establish robust BRCA1-BARD1-mediated ubiquitylation with specificity, uncover multiple modes of activity modulation, and construct a truly ligase-null variant and a variant specifically impaired in targeting nucleosomal histones. Cells expressing either of these BRCA1-BARD1 separation-of-function alleles are hypersensitive to DNA-damaging agents. Furthermore, we demonstrate that BRCA1-BARD1 ligase is not only required for DNA resection during homology-directed repair (HDR) but also contributes to later stages for HDR completion. Altogether, our findings reveal crucial, previously unrecognized roles of BRCA1-BARD1 ligase activity in genome repair via HDR, settle prior controversies regarding BRCA1-BARD1 ligase functions, and catalyze new efforts to uncover substrates related to tumor suppression.
Asunto(s)
Neoplasias , Proteínas Supresoras de Tumor , Humanos , Proteínas Supresoras de Tumor/metabolismo , Proteína BRCA1/metabolismo , Ubiquitinación , Histonas/genética , Histonas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Reparación del ADN por Recombinación , ADN , Reparación del ADNRESUMEN
Homologous recombination (HR) mediates the exchange of genetic information between sister or homologous chromatids. During HR, members of the RecA/Rad51 family of recombinases must somehow search through vast quantities of DNA sequence to align and pair single-strand DNA (ssDNA) with a homologous double-strand DNA (dsDNA) template. Here, we use single-molecule imaging to visualize Rad51 as it aligns and pairs homologous DNA sequences in real time. We show that Rad51 uses a length-based recognition mechanism while interrogating dsDNA, enabling robust kinetic selection of 8-nucleotide (nt) tracts of microhomology, which kinetically confines the search to sites with a high probability of being a homologous target. Successful pairing with a ninth nucleotide coincides with an additional reduction in binding free energy, and subsequent strand exchange occurs in precise 3-nt steps, reflecting the base triplet organization of the presynaptic complex. These findings provide crucial new insights into the physical and evolutionary underpinnings of DNA recombination.
Asunto(s)
Recombinación Homóloga , Recombinasa Rad51/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Emparejamiento Cromosómico , Reparación del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Saccharomyces cerevisiae/enzimología , Alineación de SecuenciaRESUMEN
Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in subsets of aggressive cancer. Recent studies have revealed that telomere repeat-containing RNA (TERRA) promotes ALT-associated HDR (ALT-HDR). Here, we report that RAD51AP1, a crucial ALT factor, interacts with TERRA and utilizes it to generate D- and R-loop HR intermediates. We also show that RAD51AP1 binds to and might stabilize TERRA-containing R-loops as RAD51AP1 depletion reduces R-loop formation at telomere DNA breaks. Proteomic analyses uncover a role for RAD51AP1-mediated TERRA R-loop homeostasis in a mechanism of chromatin-directed suppression of TERRA and prevention of transcription-replication collisions (TRCs) during ALT-HDR. Intriguingly, we find that both TERRA binding and this non-canonical function of RAD51AP1 require its intrinsic SUMO-SIM regulatory axis. These findings provide insights into the multi-contextual functions of RAD51AP1 within the ALT mechanism and regulation of TERRA.
Asunto(s)
ARN Largo no Codificante , Homeostasis del Telómero , Cromatina/genética , Proteómica , Telómero/genética , Telómero/metabolismo , ARN Largo no Codificante/genética , HomeostasisRESUMEN
Break-induced telomere synthesis (BITS) is a RAD51-independent form of break-induced replication that contributes to alternative lengthening of telomeres1,2. This homology-directed repair mechanism utilizes a minimal replisome comprising proliferating cell nuclear antigen (PCNA) and DNA polymerase-δ to execute conservative DNA repair synthesis over many kilobases. How this long-tract homologous recombination repair synthesis responds to complex secondary DNA structures that elicit replication stress remains unclear3-5. Moreover, whether the break-induced replisome orchestrates additional DNA repair events to ensure processivity is also unclear. Here we combine synchronous double-strand break induction with proteomics of isolated chromatin segments (PICh) to capture the telomeric DNA damage response proteome during BITS1,6. This approach revealed a replication stress-dominated response, highlighted by repair synthesis-driven DNA damage tolerance signalling through RAD18-dependent PCNA ubiquitination. Furthermore, the SNM1A nuclease was identified as the major effector of ubiquitinated PCNA-dependent DNA damage tolerance. SNM1A recognizes the ubiquitin-modified break-induced replisome at damaged telomeres, and this directs its nuclease activity to promote resection. These findings show that break-induced replication orchestrates resection-dependent lesion bypass, with SNM1A nuclease activity serving as a critical effector of ubiquitinated PCNA-directed recombination in mammalian cells.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Replicación del ADN , Recombinación Homóloga , Telómero , Moldes Genéticos , Animales , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , ADN Polimerasa III/metabolismo , Proteínas de Unión al ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Mamíferos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteómica , Recombinasa Rad51/metabolismo , Telómero/genética , Telómero/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN , Recombinación Homóloga , Complejos Multiproteicos , Humanos , Microscopía por Crioelectrón , Replicación del ADN , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/ultraestructura , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Neoplasias/genética , Nucleoproteínas/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , Recombinasa Rad51/ultraestructura , Especificidad por SustratoRESUMEN
Homologous recombination (HR) is essential for maintenance of genome integrity. Rad51 paralogs fulfill a conserved but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single-molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57 promotes assembly of Rad51 recombinase filament through transient interactions, providing evidence that it acts like a classical molecular chaperone. Srs2 is an ATP-dependent anti-recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55-Rad57 does not physically block the movement of Srs2. Instead, Rad55-Rad57 promotes rapid re-assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and single-stranded DNA (ssDNA)-bound states, the rate of which is controlled dynamically though the opposing actions of Rad55-Rad57 and Srs2.
Asunto(s)
Adenosina Trifosfatasas/genética , ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Regulación Fúngica de la Expresión Génica , Recombinación Homóloga , Recombinasa Rad51/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/metabolismo , Sitios de Unión , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Unión Proteica , Recombinasa Rad51/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Imagen Individual de Molécula , Proteína Fluorescente RojaRESUMEN
The BRCA1-BRCA2-RAD51 axis is essential for homologous recombination repair (HRR) and is frequently disrupted in breast cancers. PARP inhibitors (PARPis) are used clinically to treat BRCA-mutated breast tumors. Using a genetic screen, we identified EMI1 as a modulator of PARPi sensitivity in triple-negative breast cancer (TNBC) cells. This function requires the F-box domain of EMI1, through which EMI1 assembles a canonical SCF ubiquitin ligase complex that constitutively targets RAD51 for degradation. In response to genotoxic stress, CHK1-mediated phosphorylation of RAD51 counteracts EMI1-dependent degradation by enhancing RAD51's affinity for BRCA2, leading to RAD51 accumulation. Inhibition of RAD51 degradation restores HRR in BRCA1-depleted cells. Human breast cancer samples display an inverse correlation between EMI1 and RAD51 protein levels. A subset of BRCA1-deficient TNBC cells develop resistance to PARPi by downregulating EMI1 and restoring RAD51-dependent HRR. Notably, reconstitution of EMI1 expression reestablishes PARPi sensitivity both in cellular systems and in an orthotopic mouse model.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Resistencia a Antineoplásicos , Proteínas F-Box/metabolismo , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Daño del ADN , Resistencia a Antineoplásicos/genética , Proteínas F-Box/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Ratones Endogámicos NOD , Ratones SCID , Fosforilación , Proteolisis , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Reparación del ADN por Recombinación , Transducción de Señal/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The vast majority of eukaryotes possess two DNA recombinases: Rad51, which is ubiquitously expressed, and Dmc1, which is meiosis-specific. The evolutionary origins of this two-recombinase system remain poorly understood. Interestingly, Dmc1 can stabilize mismatch-containing base triplets, whereas Rad51 cannot. Here, we demonstrate that this difference can be attributed to three amino acids conserved only within the Dmc1 lineage of the Rad51/RecA family. Chimeric Rad51 mutants harboring Dmc1-specific amino acids gain the ability to stabilize heteroduplex DNA joints with mismatch-containing base triplets, whereas Dmc1 mutants with Rad51-specific amino acids lose this ability. Remarkably, RAD-51 from Caenorhabditis elegans, an organism without Dmc1, has acquired "Dmc1-like" amino acids. Chimeric C. elegans RAD-51 harboring "canonical" Rad51 amino acids gives rise to toxic recombination intermediates, which must be actively dismantled to permit normal meiotic progression. We propose that Dmc1 lineage-specific amino acids involved in the stabilization of heteroduplex DNA joints with mismatch-containing base triplets may contribute to normal meiotic recombination.
Asunto(s)
Aminoácidos/metabolismo , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , Recombinasas/química , Recombinasas/metabolismo , Recombinación Genética/genética , Aminoácidos/genética , Animales , Disparidad de Par Base , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Secuencia Conservada , Mutación , Recombinasa Rad51/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Recombinasas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
In this issue of Molecular Cell, Mazina et al. (2017) describe how the Rad52 protein mediates RNA-dependent DNA double-strand break repair via inverse strand exchange. This finding sheds light on how eukaryotes utilize RNA to repair chromosome breaks.
Asunto(s)
Reparación del ADN , Recombinasa Rad51/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/genética , Proteína Recombinante y Reparadora de ADN Rad52/genética , Recombinación Genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Environmental agents like ionizing radiation (IR) and chemotherapeutic drugs can cause severe damage to the DNA, often in the form of double-strand breaks (DSBs). Remaining unrepaired, DSBs can lead to chromosomal rearrangements, and cell death. One major error-free pathway to repair DSBs is homologous recombination repair (HRR). Tousled-like kinase 1 (TLK1), a Ser/Thr kinase that regulates the DNA damage checkpoint, has been found to interact with RAD54, a central DNA translocase in HRR. To determine how TLK1 regulates RAD54, we inhibited or depleted TLK1 and tested how this impacts HRR in human cells using a ISce-I-GR-DsRed fused reporter endonuclease. Our results show that TLK1 phosphorylates RAD54 at three threonines (T41, T59 and T700), two of which are located within its N-terminal domain (NTD) and one is located within its C-terminal domain (CTD). Phosphorylation at both T41 and T59 supports HRR and protects cells from DNA DSB damage. In contrast, phosphorylation of T700 leads to impaired HRR and engenders no protection to cells from cytotoxicity and rather results in repair delay. Further, our work enlightens the effect of RAD54-T700 (RAD54-CTD) phosphorylation by TLK1 in mammalian system and reveals a new site of interaction with RAD51.
Asunto(s)
Reparación del ADN , Reparación del ADN por Recombinación , Animales , Humanos , Fosforilación , Daño del ADN , ADN/metabolismo , Recombinasa Rad51/metabolismo , Recombinación Homóloga , Mamíferos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Srs2 is a superfamily 1 (SF1) helicase that participates in several pathways necessary for the repair of damaged DNA. Srs2 regulates formation of early homologous recombination (HR) intermediates by actively removing the recombinase Rad51 from single-stranded DNA (ssDNA). It is not known whether and how Srs2 itself is down-regulated to allow for timely HR progression. Rad54 and Rdh54 are two closely related superfamily 2 (SF2) motor proteins that promote the formation of Rad51-dependent recombination intermediates. Rad54 and Rdh54 bind tightly to Rad51-ssDNA and act downstream of Srs2, suggesting that they may affect the ability of Srs2 to dismantle Rad51 filaments. Here, we used DNA curtains to determine whether Rad54 and Rdh54 alter the ability of Srs2 to disrupt Rad51 filaments. We show that Rad54 and Rdh54 act synergistically to greatly restrict the antirecombinase activity of Srs2. Our findings suggest that Srs2 may be accorded only a limited time window to act and that Rad54 and Rdh54 fulfill a role of prorecombinogenic licensing factors.
Asunto(s)
ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/metabolismo , ADN-Topoisomerasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Daño del ADN/fisiología , ADN Helicasas/fisiología , Reparación del ADN/genética , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/fisiología , ADN-Topoisomerasas/fisiología , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Recombinación Homóloga/genética , Unión Proteica/genética , Recombinasa Rad51/metabolismo , Recombinasa Rad51/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologíaRESUMEN
The budding yeast Mre11-Rad50-Xrs2 (MRX) complex and Sae2 function together in DNA end resection during homologous recombination. Here we show that the Ku complex shields DNA ends from exonucleolytic digestion but facilitates endonucleolytic scission by MRX with a dependence on ATP and Sae2. The incision site is enlarged into a DNA gap via the exonuclease activity of MRX, which is stimulated by Sae2 without ATP being present. RPA renders a partially resected or palindromic DNA structure susceptible to MRX-Sae2, and internal protein blocks also trigger DNA cleavage. We present models for how MRX-Sae2 creates entry sites for the long-range resection machinery.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Reparación del ADN/fisiología , Endonucleasas/metabolismo , Exonucleasas/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , División del ADN , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Activación Enzimática/genética , Exodesoxirribonucleasas/metabolismo , Complejos Multiproteicos/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Pif1 is a broadly conserved helicase that is essential for genome integrity and participates in numerous aspects of DNA metabolism, including telomere length regulation, Okazaki fragment maturation, replication fork progression through difficult-to-replicate sites, replication fork convergence, and break-induced replication. However, details of its translocation properties and the importance of amino acids residues implicated in DNA binding remain unclear. Here, we use total internal reflection fluorescence microscopy with single-molecule DNA curtain assays to directly observe the movement of fluorescently tagged Saccharomyces cerevisiae Pif1 on single-stranded DNA (ssDNA) substrates. We find that Pif1 binds tightly to ssDNA and translocates very rapidly (â¼350 nucleotides per second) in the 5'â3' direction over relatively long distances (â¼29,500 nucleotides). Surprisingly, we show the ssDNA-binding protein replication protein A inhibits Pif1 activity in both bulk biochemical and single-molecule measurements. However, we demonstrate Pif1 can strip replication protein A from ssDNA, allowing subsequent molecules of Pif1 to translocate unimpeded. We also assess the functional attributes of several Pif1 mutations predicted to impair contact with the ssDNA substrate. Taken together, our findings highlight the functional importance of these amino acid residues in coordinating the movement of Pif1 along ssDNA.
Asunto(s)
ADN de Cadena Simple , Proteínas de Saccharomyces cerevisiae , ADN/metabolismo , Replicación del ADN , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Nucleótidos/metabolismo , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Rad54 and Rdh54 are closely related ATP-dependent motor proteins that participate in homologous recombination (HR). During HR, these enzymes functionally interact with the Rad51 presynaptic complex (PSC). Despite their importance, we know little about how they are organized within the PSC, or how their organization affects PSC function. Here, we use single-molecule optical microscopy and genetic analysis of chimeric protein constructs to evaluate the binding distributions of Rad54 and Rdh54 within the PSC. We find that Rad54 and Rdh54 have distinct binding sites within the PSC, which allow these proteins to act cooperatively as DNA sequences are aligned during homology search. Our data also reveal that Rad54 must bind to a specific location within the PSC, whereas Rdh54 retains its function in the repair of MMS-induced DNA damage even when recruited to the incorrect location. These findings support a model in which the relative binding sites of Rad54 and Rdh54 help to define their functions during mitotic HR.
Asunto(s)
Emparejamiento Cromosómico , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/metabolismo , ADN-Topoisomerasas/metabolismo , ADN de Cadena Simple/metabolismo , Recombinasa Rad51/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Dominio Catalítico/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN Helicasas/genética , Reparación del ADN/genética , Enzimas Reparadoras del ADN/genética , ADN-Topoisomerasas/genética , Proteínas de Unión al ADN/metabolismo , Mutación , Unión Proteica , Dominios Proteicos , Recombinasa Rad51/genética , Proteínas Recombinantes , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The tumour suppressor complex BRCA1-BARD1 functions in the repair of DNA double-stranded breaks by homologous recombination. During this process, BRCA1-BARD1 facilitates the nucleolytic resection of DNA ends to generate a single-stranded template for the recruitment of another tumour suppressor complex, BRCA2-PALB2, and the recombinase RAD51. Here, by examining purified wild-type and mutant BRCA1-BARD1, we show that both BRCA1 and BARD1 bind DNA and interact with RAD51, and that BRCA1-BARD1 enhances the recombinase activity of RAD51. Mechanistically, BRCA1-BARD1 promotes the assembly of the synaptic complex, an essential intermediate in RAD51-mediated DNA joint formation. We provide evidence that BRCA1 and BARD1 are indispensable for RAD51 stimulation. Notably, BRCA1-BARD1 mutants with weakened RAD51 interactions show compromised DNA joint formation and impaired mediation of homologous recombination and DNA repair in cells. Our results identify a late role of BRCA1-BARD1 in homologous recombination, an attribute of the tumour suppressor complex that could be targeted in cancer therapy.