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.
Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded ...DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly.
Crops lack genetic resistance to most necrotrophic pathogens. To compensate for this disadvantage, plants recruit antagonistic members of the soil microbiome to defend their roots against pathogens ...and other pests. The best examples of this microbially based defense of roots are observed in disease-suppressive soils in which suppressiveness is induced by continuously growing crops that are susceptible to a pathogen, but the molecular basis of most is poorly understood. Here we report the microbial characterization of a Korean soil with specific suppressiveness to Fusarium wilt of strawberry. In this soil, an attack on strawberry roots by Fusarium oxysporum results in a response by microbial defenders, of which members of the Actinobacteria appear to have a key role. We also identify Streptomyces genes responsible for the ribosomal synthesis of a novel heat-stable antifungal thiopeptide antibiotic inhibitory to F. oxysporum and the antibiotic's mode of action against fungal cell wall biosynthesis. Both classical- and community-oriented approaches were required to dissect this suppressive soil from the field to the molecular level, and the results highlight the role of natural antibiotics as weapons in the microbial warfare in the rhizosphere that is integral to plant health, vigor and development.
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.
Bloom syndrome (BS) is associated with a profoundly increased cancer risk and is caused by mutations in the Bloom helicase (BLM). BLM is involved in the nucleolytic processing of the ends of DNA ...double-strand breaks (DSBs), to yield long 3' ssDNA tails that serve as the substrate for break repair by homologous recombination (HR). Here, we use single-molecule imaging to demonstrate that BLM mediates formation of large ssDNA loops during DNA end processing. A BLM mutant lacking the N-terminal domain (NTD) retains vigorous in vitro end processing activity but fails to generate ssDNA loops. This same mutant supports DSB end processing in cells, however, these cells do not form RAD51 DNA repair foci and the processed DSBs are channeled into synthesis-dependent strand annealing (SSA) instead of HR-mediated repair, consistent with a defect in RAD51 filament formation. Together, our results provide insights into BLM functions during homologous recombination.
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.
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•Single-molecule studies of Rad55-Rad57 mechanism of action•Rad55-Rad57 binds Rad51-ssDNA during filament assembly and quickly dissociates•The anti-recombinase Srs2 is not physically blocked by Rad55-Rad57•Rad55-Rad57 stimulates rapid Rad51 filament reassembly after passage of Srs2
Roy et al. present a single-molecule analysis of the Rad51 paralog complex Rad55-Rad57. They show that Rad55-Rad57 binds transiently to Rad51-ssDNA to promote Rad51 filament assembly but then dissociates quickly as the filaments mature. They further demonstrate that Rad55-Rad57 does not block the translocase Srs2.
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.
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•EMI1 assembles an SCF ubiquitin ligase that constitutively targets RAD51 for degradation•In response to genotoxic stress, CHK1 counteracts EMI1-dependent degradation of RAD51•A subset of TNBC cells develop resistance to PARPi by downregulating EMI1•Reconstitution of EMI1 expression reestablishes PARPi sensitivity in vitro and in vivo
Marzio et al. report that cellular levels of RAD51 are kept in check by EMI1-mediated degradation. Upon DNA damage, CHK1 phosphorylation of RAD51 on Thr309 counteracts this event, allowing RAD51 accumulation and HRR. Downregulation of EMI1 in BRCA1-deficient breast cancer cells induces primary and acquired resistance to PARPi both in vitro and in vivo.
DNA strand exchange plays a central role in genetic recombination across all kingdoms of life, but the physical basis for these reactions remains poorly defined. Using single-molecule imaging, we ...found that bacterial RecA and eukaryotic Rad51 and Dmc1 all stabilize strand exchange intermediates in precise three-nucleotide steps. Each step coincides with an energetic signature (0.3 kBT) that is conserved from bacteria to humans. Triplet recognition is strictly dependent on correct Watson-Crick pairing. Rad51, RecA, and Dmc1 can all step over mismatches, but only Dmc1 can stabilize mismatched triplets. This finding provides insight into why eukaryotes have evolved a meiosis-specific recombinase. We propose that canonical Watson-Crick base triplets serve as the fundamental unit of pairing interactions during DNA recombination.
Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein necessary for all aspects of DNA metabolism involving an ssDNA intermediate, including DNA ...replication, repair, recombination, DNA damage response and checkpoint activation, and telomere maintenance. The role of RPA in most of these reactions is to protect the ssDNA until it can be delivered to downstream enzymes. Therefore a crucial feature of RPA is that it must bind very tightly to ssDNA, but must also be easily displaced from ssDNA to allow other proteins to gain access to the substrate. Here we use total internal reflection fluorescence microscopy and nanofabricated DNA curtains to visualize the behavior of Saccharomyces cerevisiae RPA on individual strands of ssDNA in real-time. Our results show that RPA remains bound to ssDNA for long periods of time when free protein is absent from solution. In contrast, RPA rapidly dissociates from ssDNA when free RPA is present in solution allowing rapid exchange between the free and bound states. In addition, the S. cerevisiae DNA recombinase Rad51 and E. coli single-stranded binding protein (SSB) also promote removal of RPA from ssDNA. These results reveal an unanticipated exchange between bound and free RPA suggesting a binding mechanism that can confer exceptionally slow off rates, yet also enables rapid displacement through a direct exchange mechanism that is reliant upon the presence of free ssDNA-binding proteins in solution. Our results indicate that RPA undergoes constant microscopic dissociation under all conditions, but this is only manifested as macroscopic dissociation (i.e. exchange) when free proteins are present in solution, and this effect is due to mass action. We propose that the dissociation of RPA from ssDNA involves a partially dissociated intermediate, which exposes a small section of ssDNA allowing other proteins to access to the DNA.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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