RESUMO
In Saccharomyces cerevisiae (S. cerevisiae), Mre11-Rad50-Xrs2 (MRX)-Sae2 nuclease activity is required for the resection of DNA breaks with secondary structures or protein blocks, while in humans, the MRE11-RAD50-NBS1 (MRN) homolog with CtIP is needed to initiate DNA end resection of all breaks. Phosphorylated Sae2/CtIP stimulates the endonuclease activity of MRX/N. Structural insights into the activation of the Mre11 nuclease are available only for organisms lacking Sae2/CtIP, so little is known about how Sae2/CtIP activates the nuclease ensemble. Here, we uncover the mechanism of Mre11 activation by Sae2 using a combination of AlphaFold2 structural modeling of biochemical and genetic assays. We show that Sae2 stabilizes the Mre11 nuclease in a conformation poised to cleave substrate DNA. Several designs of compensatory mutations establish how Sae2 activates MRX in vitro and in vivo, supporting the structural model. Finally, our study uncovers how human CtIP, despite considerable sequence divergence, employs a similar mechanism to activate MRN.
Assuntos
Proteínas de Ligação a DNA , Endodesoxirribonucleases , Endonucleases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Endonucleases/metabolismo , Endonucleases/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/química , Humanos , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Modelos Moleculares , Fosforilação , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Quebras de DNA de Cadeia Dupla , Hidrolases Anidrido Ácido/metabolismo , Hidrolases Anidrido Ácido/genética , Mutação , Proteína Homóloga a MRE11/metabolismo , Proteína Homóloga a MRE11/genética , Reparo do DNA , Ativação EnzimáticaRESUMO
G-quadruplexes (G4s) form throughout the genome and influence important cellular processes. Their deregulation can challenge DNA replication fork progression and threaten genome stability. Here, we demonstrate an unexpected role for the double-stranded DNA (dsDNA) translocase helicase-like transcription factor (HLTF) in responding to G4s. We show that HLTF, which is enriched at G4s in the human genome, can directly unfold G4s in vitro and uses this ATP-dependent translocase function to suppress G4 accumulation throughout the cell cycle. Additionally, MSH2 (a component of MutS heterodimers that bind G4s) and HLTF act synergistically to suppress G4 accumulation, restrict alternative lengthening of telomeres, and promote resistance to G4-stabilizing drugs. In a discrete but complementary role, HLTF restrains DNA synthesis when G4s are stabilized by suppressing primase-polymerase (PrimPol)-dependent repriming. Together, the distinct roles of HLTF in the G4 response prevent DNA damage and potentially mutagenic replication to safeguard genome stability.
RESUMO
DNA double-strand breaks (DSBs) are cytotoxic genome lesions that must be accurately and efficiently repaired to ensure genome integrity. In yeast, the Mre11-Rad50-Xrs2 (MRX) complex nicks 5'-terminated DSB ends to initiate nucleolytic processing of DSBs for repair by homologous recombination. How MRX-DNA interactions support 5' strand-specific nicking and how nicking is influenced by the chromatin context have remained elusive. Using a deep sequencing-based assay, we mapped MRX nicks at single-nucleotide resolution next to multiple DSBs in the yeast genome. We observed that the DNA end-binding Ku70-Ku80 complex directed DSB-proximal nicks and that repetitive MRX cleavage extended the length of resection tracts. We identified a sequence motif and a DNA meltability profile that is preferentially nicked by MRX. Furthermore, we found that nucleosomes as well as transcription impeded MRX incisions. Our findings suggest that local DNA sequence and chromatin features shape the activity of this central DSB repair complex.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromatina/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Reparo do DNA , DNA/genéticaRESUMO
DNA double-strand break (DSB) repair is initiated by DNA end resection. CtIP acts in short-range resection to stimulate MRE11-RAD50-NBS1 (MRN) to endonucleolytically cleave 5'-terminated DNA to bypass protein blocks. CtIP also promotes the DNA2 helicase-nuclease to accelerate long-range resection downstream from MRN. Here, using AlphaFold2, we identified CtIP-F728E-Y736E as a separation-of-function mutant that is still proficient in conjunction with MRN but is not able to stimulate ssDNA degradation by DNA2. Accordingly, CtIP-F728E-Y736E impairs physical interaction with DNA2. Cellular assays revealed that CtIP-F728E-Y736E cells exhibit reduced DSB-dependent chromatin-bound RPA, impaired long-range resection, and increased sensitivity to DSB-inducing drugs. Previously, CtIP was shown to be targeted by PLK1 to inhibit long-range resection, yet the underlying mechanism was unclear. We show that the DNA2-interacting region in CtIP includes the PLK1 target site at S723. The integrity of S723 in CtIP is necessary for the stimulation of DNA2, and phosphorylation of CtIP by PLK1 in vitro is consequently inhibitory, explaining why PLK1 restricts long-range resection. Our data support a model in which CDK-dependent phosphorylation of CtIP activates resection by MRN in S phase, and PLK1-mediated phosphorylation of CtIP disrupts CtIP stimulation of DNA2 to attenuate long-range resection later at G2/M.
Assuntos
Proteínas de Transporte , Quebras de DNA de Cadeia Dupla , Proteínas de Transporte/genética , Endodesoxirribonucleases/metabolismo , Reparo do DNA , DNA Helicases/genética , DNA Helicases/metabolismo , DNARESUMO
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
DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genome integrity and cell viability. Typically, cells repair DSBs by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The relative use of these two pathways depends on many factors, including cell cycle stage and the nature of the DNA ends. A critical determinant of repair pathway selection is the initiation of 5'â3' nucleolytic degradation of DNA ends, a process referred to as DNA end resection. End resection is essential to create single-stranded DNA overhangs, which serve as the substrate for the Rad51 recombinase to initiate HR and are refractory to NHEJ repair. Here, we review recent insights into the mechanisms of end resection, how it is regulated, and the pathological consequences of its dysregulation.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , DNA , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Recombinação Homóloga/genéticaRESUMO
In this issue of Molecular Cell, Roy et al. (2021) and Belan et al. (2021) demonstrate that the yeast and nematode RAD51 paralog complexes function as chaperones to promote the assembly of the RAD51 nucleoprotein filament on RPA-coated ssDNA.
Assuntos
Rad51 Recombinase , Proteínas de Saccharomyces cerevisiae , DNA de Cadeia Simples/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de MoléculaRESUMO
Crossover recombination is essential for accurate chromosome segregation during meiosis. The MutSγ complex, Msh4-Msh5, facilitates crossing over by binding and stabilizing nascent recombination intermediates. We show that these activities are governed by regulated proteolysis. MutSγ is initially inactive for crossing over due to an N-terminal degron on Msh4 that renders it unstable by directly targeting proteasomal degradation. Activation of MutSγ requires the Dbf4-dependent kinase Cdc7 (DDK), which directly phosphorylates and thereby neutralizes the Msh4 degron. Genetic requirements for Msh4 phosphorylation indicate that DDK targets MutSγ only after it has bound to nascent joint molecules (JMs) in the context of synapsing chromosomes. Overexpression studies confirm that the steady-state level of Msh4, not phosphorylation per se, is the critical determinant for crossing over. At the DNA level, Msh4 phosphorylation enables the formation and crossover-biased resolution of double-Holliday Junction intermediates. Our study establishes regulated protein degradation as a fundamental mechanism underlying meiotic crossing over.
Assuntos
Troca Genética , Proteínas de Ligação a DNA/metabolismo , Meiose/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Pareamento Cromossômico , Proteínas de Ligação a DNA/química , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
The Werner Syndrome helicase, WRN, is a promising therapeutic target in cancers with microsatellite instability (MSI). Long-term MSI leads to the expansion of TA nucleotide repeats proposed to form cruciform DNA structures, which in turn cause DNA breaks and cell lethality upon WRN downregulation. Here we employed biochemical assays to show that WRN helicase can efficiently and directly unfold cruciform structures, thereby preventing their cleavage by the SLX1-SLX4 structure-specific endonuclease. TA repeats are particularly prone to form cruciform structures, explaining why these DNA sequences are preferentially broken in MSI cells upon WRN downregulation. We further demonstrate that the activity of the DNA mismatch repair (MMR) complexes MutSα (MSH2-MSH6), MutSß (MSH2-MSH3), and MutLα (MLH1-PMS2) similarly decreases the level of DNA cruciforms, although the mechanism is different from that employed by WRN. When combined, WRN and MutLα exhibited higher than additive effects in in vitro cruciform processing, suggesting that WRN and the MMR proteins may cooperate. Our data explain how WRN and MMR defects cause genome instability in MSI cells with expanded TA repeats, and provide a mechanistic basis for their recently discovered synthetic-lethal interaction with promising applications in precision cancer therapy.
Assuntos
Reparo de Erro de Pareamento de DNA , DNA Cruciforme , Humanos , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Instabilidade de Microssatélites , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismo , Proteína 1 Homóloga a MutL/genéticaRESUMO
During prophase of the first meiotic division, cells deliberately break their DNA1. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes2. A pathway that depends on the MLH1-MLH3 (MutLγ) nuclease has been implicated in the biased processing of meiotic recombination intermediates into crossovers by an unknown mechanism3-7. Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4-MSH5 (MutSγ), which supports crossing over8, binds branched recombination intermediates and associates with MutLγ, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutSγ directly stimulates DNA cleavage by the MutLγ endonuclease. MutLγ activity is further stimulated by EXO1, but only when MutSγ is present. Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are additional components of the nuclease ensemble, thereby triggering crossing-over. Saccharomyces cerevisiae strains in which MutLγ cannot interact with PCNA present defects in forming crossovers. Finally, the MutLγ-MutSγ-EXO1-RFC-PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, it probably processes meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points9. As DNA nicking by MutLγ depends on its co-factors, the asymmetric distribution of MutSγ and RFC-PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This mode of MutLγ nuclease activation might explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes4.
Assuntos
Troca Genética , Endonucleases/metabolismo , Meiose , Proteína 1 Homóloga a MutL/metabolismo , Proteínas MutL/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Cromossomos Humanos/genética , Sequência Conservada , DNA/metabolismo , Clivagem do DNA , Enzimas Reparadoras do DNA/metabolismo , DNA Cruciforme/metabolismo , Exodesoxirribonucleases/metabolismo , Humanos , Proteína 1 Homóloga a MutL/química , Proteínas MutL/química , Proteínas MutS/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína de Replicação C/metabolismoRESUMO
Meiotic crossover formation requires the stabilization of early recombination intermediates by a set of proteins and occurs within the environment of the chromosome axis, a structure important for the regulation of meiotic recombination events. The molecular mechanisms underlying and connecting crossover recombination and axis localization are elusive. Here, we identified the ZZS (Zip2-Zip4-Spo16) complex, required for crossover formation, which carries two distinct activities: one provided by Zip4, which acts as hub through physical interactions with components of the chromosome axis and the crossover machinery, and the other carried by Zip2 and Spo16, which preferentially bind branched DNA molecules in vitro. We found that Zip2 and Spo16 share structural similarities to the structure-specific XPF-ERCC1 nuclease, although it lacks endonuclease activity. The XPF domain of Zip2 is required for crossover formation, suggesting that, together with Spo16, it has a noncatalytic DNA recognition function. Our results suggest that the ZZS complex shepherds recombination intermediates toward crossovers as a dynamic structural module that connects recombination events to the chromosome axis. The identification of the ZZS complex improves our understanding of the various activities required for crossover implementation and is likely applicable to other organisms, including mammals.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Troca Genética , Proteínas de Ligação a DNA/metabolismo , Meiose/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Cromossômicas não Histona/química , Cromossomos Fúngicos , DNA/química , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/química , Endodesoxirribonucleases/metabolismo , Proteínas Associadas aos Microtúbulos/química , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/químicaAssuntos
Neoplasias , Humanos , Neoplasias/genética , Neoplasias/terapia , Reparo do DNA , DNA , Dano ao DNARESUMO
To ensure the completion of DNA replication and maintenance of genome integrity, DNA repair factors protect stalled replication forks upon replication stress. Previous studies have identified a critical role for the tumor suppressors BRCA1 and BRCA2 in preventing the degradation of nascent DNA by the MRE11 nuclease after replication stress. Here we show that depletion of SMARCAL1, a SNF2-family DNA translocase that remodels stalled forks, restores replication fork stability and reduces the formation of replication stress-induced DNA breaks and chromosomal aberrations in BRCA1/2-deficient cells. In addition to SMARCAL1, other SNF2-family fork remodelers, including ZRANB3 and HLTF, cause nascent DNA degradation and genomic instability in BRCA1/2-deficient cells upon replication stress. Our observations indicate that nascent DNA degradation in BRCA1/2-deficient cells occurs as a consequence of MRE11-dependent nucleolytic processing of reversed forks generated by fork remodelers. These studies provide mechanistic insights into the processes that cause genome instability in BRCA1/2-deficient cells.
Assuntos
Proteína BRCA2/deficiência , Quebras de DNA , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Linhagem Celular Tumoral , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica , Humanos , Proteína Homóloga a MRE11 , Fatores de Transcrição/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
DNA double-strand break repair by homologous recombination is initiated by DNA end resection, which is commenced by the Mre11-Rad50-Xrs2 complex and Sae2 in yeast. Here we report that the nonhomologous end joining factor Ku limits the exonuclease activity of Mre11 and promotes its endonuclease to cleave 5'-terminated DNA strands at break sites. Following initial endonucleolytic cleavage past the obstacle, Exo1 specifically extends the resection track, leading to the generation of long 3' overhangs that are required for homologous recombination. These experiments provide mechanistic insights into how short-range and long-range DNA end resection enzymes overcome obstacles near broken DNA ends to initiate recombination.
Assuntos
Reparo do DNA por Junção de Extremidades , Endonucleases/metabolismo , Exonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Animais , Clivagem do DNA , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Ativação Enzimática/genética , Exodesoxirribonucleases/metabolismo , Complexos Multiproteicos/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Células Sf9RESUMO
DNA double strand breaks (DSBs) can be detrimental to the cell and need to be efficiently repaired. A first step in DSB repair is to bring the free ends in close proximity to enable ligation by non-homologous end-joining (NHEJ), while the more precise, but less available, repair by homologous recombination (HR) requires close proximity of a sister chromatid. The human MRE11-RAD50-NBS1 (MRN) complex, Mre11-Rad50-Xrs2 (MRX) in yeast, is involved in both repair pathways. Here we use nanofluidic channels to study, on the single DNA molecule level, how MRN, MRX and their constituents interact with long DNA and promote DNA bridging. Nanofluidics is a suitable method to study reactions on DNA ends since no anchoring of the DNA end(s) is required. We demonstrate that NBS1 and Xrs2 play important, but differing, roles in the DNA tethering by MRN and MRX. NBS1 promotes DNA bridging by MRN consistent with tethering of a repair template. MRX shows a "synapsis-like" DNA end-bridging, stimulated by the Xrs2 subunit. Our results highlight the different ways MRN and MRX bridge DNA, and the results are in agreement with their key roles in HR and NHEJ, respectively, and contribute to the understanding of the roles of NBS1 and Xrs2 in DSB repair.
Assuntos
Proteínas de Ligação a DNA , Endodesoxirribonucleases , Proteínas de Saccharomyces cerevisiae , Humanos , DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
To repair a DNA double-strand break (DSB) by homologous recombination (HR), the 5'-terminated strand of the DSB must be resected. The human MRE11-RAD50-NBS1 (MRN) and CtIP proteins were implicated in the initiation of DNA end resection, but the underlying mechanism remained undefined. Here, we show that CtIP is a co-factor of the MRE11 endonuclease activity within the MRN complex. This function is absolutely dependent on CtIP phosphorylation that includes the key cyclin-dependent kinase target motif at Thr-847. Unlike in yeast, where the Xrs2/NBS1 subunit is dispensable in vitro, NBS1 is absolutely required in the human system. The MRE11 endonuclease in conjunction with RAD50, NBS1, and phosphorylated CtIP preferentially cleaves 5'-terminated DNA strands near DSBs. Our results define the initial step of HR that is particularly relevant for the processing of DSBs bearing protein blocks.
Assuntos
Proteínas de Ciclo Celular/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Helicases/genética , Recombinação Homóloga/genética , Complexos Multiproteicos/genética , Hidrolases Anidrido Ácido , Proteínas de Transporte , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA , Endodesoxirribonucleases , Humanos , Proteína Homóloga a MRE11 , Proteínas Nucleares , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex orchestrates the cellular response to DSBs through its structural, enzymatic, and signaling roles. Xrs2/Nbs1 is essential for nuclear translocation of Mre11, but its role as a component of the complex is not well defined. Here, we demonstrate that nuclear localization of Mre11 (Mre11-NLS) is able to bypass several functions of Xrs2, including DNA end resection, meiosis, hairpin resolution, and cellular resistance to clastogens. Using purified components, we show that the MR complex has equivalent activity to MRX in cleavage of protein-blocked DNA ends. Although Xrs2 physically interacts with Sae2, we found that end resection in its absence remains Sae2 dependent in vivo and in vitro. MRE11-NLS was unable to rescue the xrs2Δ defects in Tel1/ATM kinase signaling and non-homologous end joining, consistent with the role of Xrs2 as a chaperone and adaptor protein coordinating interactions between the MR complex and other repair proteins.
Assuntos
Reparo do DNA por Junção de Extremidades , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sítios de Ligação , Camptotecina/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/deficiência , Endonucleases/genética , Exodesoxirribonucleases/metabolismo , Regulação Fúngica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metanossulfonato de Metila/farmacologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
SMARCAL1, ZRANB3 and HLTF are required for the remodeling of replication forks upon stress to promote genome stability. RAD51, along with the RAD51 paralog complex, were also found to have recombination-independent functions in fork reversal, yet the underlying mechanisms remained unclear. Using reconstituted reactions, we build upon previous data to show that SMARCAL1, ZRANB3 and HLTF have unequal biochemical capacities, explaining why they have non-redundant functions. SMARCAL1 uniquely anneals RPA-coated ssDNA, which depends on its direct interaction with RPA, but not on ATP. SMARCAL1, along with ZRANB3, but not HLTF efficiently employ ATPase driven translocase activity to rezip RPA-covered bubbled DNA, which was proposed to mimic elements of fork reversal. In contrast, ZRANB3 and HLTF but not SMARCAL1 are efficient in branch migration that occurs downstream in fork remodeling. We also show that low concentrations of RAD51 and the RAD51 paralog complex, RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2), directly stimulate the motor-driven activities of SMARCAL1 and ZRANB3 but not HLTF, and the interplay is underpinned by physical interactions. Our data provide a possible mechanism explaining previous cellular experiments implicating RAD51 and BCDX2 in fork reversal.
Assuntos
DNA Helicases , Replicação do DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA/genética , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica , Humanos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Fatores de Transcrição/genéticaRESUMO
In budding yeast, the MutL homolog heterodimer Mlh1-Mlh3 (MutLγ) plays a central role in the formation of meiotic crossovers. It is also involved in the repair of a subset of mismatches besides the main mismatch repair (MMR) endonuclease Mlh1-Pms1 (MutLα). The heterodimer interface and endonuclease sites of MutLγ and MutLα are located in their C-terminal domain (CTD). The molecular basis of MutLγ's dual roles in MMR and meiosis is not known. To better understand the specificity of MutLγ, we characterized the crystal structure of Saccharomyces cerevisiae MutLγ(CTD). Although MutLγ(CTD) presents overall similarities with MutLα(CTD), it harbors some rearrangement of the surface surrounding the active site, which indicates altered substrate preference. The last amino acids of Mlh1 participate in the Mlh3 endonuclease site as previously reported for Pms1. We characterized mlh1 alleles and showed a critical role of this Mlh1 extreme C terminus both in MMR and in meiotic recombination. We showed that the MutLγ(CTD) preferentially binds Holliday junctions, contrary to MutLα(CTD). We characterized Mlh3 positions on the N-terminal domain (NTD) and CTD that could contribute to the positioning of the NTD close to the CTD in the context of the full-length MutLγ. Finally, crystal packing revealed an assembly of MutLγ(CTD) molecules in filament structures. Mutation at the corresponding interfaces reduced crossover formation, suggesting that these superstructures may contribute to the oligomer formation proposed for MutLγ. This study defines clear divergent features between the MutL homologs and identifies, at the molecular level, their specialization toward MMR or meiotic recombination functions.