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.
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•RAD51AP1 is a DNA-binding protein that stimulates RAD51 activity.•RAD51AP1 protects against DNA damage and is required for replication fork and genome stability.•RAD51AP1 expression ...may be linked to cancer development and progression.•RAD51AP1 has the potential to become a tumor biomarker.•RAD51AP1 may provide a new target in cancer therapy.
Homologous recombination (HR) serves to repair DNA double-strand breaks and damaged replication forks and is essential for maintaining genome stability and tumor suppression. HR capacity also determines the efficacy of anticancer therapy. Hence, there is an urgent need to better understand all HR proteins and sub-pathways. An emerging protein that is critical for RAD51-mediated HR is RAD51-associated protein 1 (RAD51AP1). Although much has been learned about its biochemical attributes, the precise molecular role of RAD51AP1 in the HR reaction is not yet fully understood. The available literature also suggests that RAD51AP1 expression may be relevant for cancer development and progression. Here, we review the efforts that led to the discovery of RAD51AP1 and elaborate on our current understanding of its biochemical profile and biological function. We also discuss how RAD51AP1 may help to promote cancer development and why it could potentially represent a promising new target for therapeutic intervention.
Fanconi anemia (FA) is a multigenic disease of bone marrow failure and cancer susceptibility stemming from a failure to remove DNA crosslinks and other chromosomal lesions. Within the FA DNA damage ...response pathway, DNA-dependent monoubiquitinaton of FANCD2 licenses downstream events, while timely FANCD2 deubiquitination serves to extinguish the response. Here, we show with reconstituted biochemical systems, which we developed, that efficient FANCD2 deubiquitination by the USP1-UAF1 complex is dependent on DNA and DNA binding by UAF1. Surprisingly, we find that the DNA binding activity of the UAF1-associated protein RAD51AP1 can substitute for that of UAF1 in FANCD2 deubiquitination in our biochemical system. We also reveal the importance of DNA binding by UAF1 and RAD51AP1 in FANCD2 deubiquitination in the cellular setting. Our results provide insights into a key step in the FA pathway and help define the multifaceted role of the USP1-UAF1-RAD51AP1 complex in DNA damage tolerance and genome repair.
The tumor suppressor BRCA2 is thought to facilitate the handoff of ssDNA from replication protein A (RPA) to the RAD51 recombinase during DNA break and replication fork repair by homologous ...recombination. However, we find that RPA-RAD51 exchange requires the BRCA2 partner DSS1. Biochemical, structural, and in vivo analyses reveal that DSS1 allows the BRCA2-DSS1 complex to physically and functionally interact with RPA. Mechanistically, DSS1 acts as a DNA mimic to attenuate the affinity of RPA for ssDNA. A mutation in the solvent-exposed acidic domain of DSS1 compromises the efficacy of RPA-RAD51 exchange. Thus, by targeting RPA and mimicking DNA, DSS1 functions with BRCA2 in a two-component homologous recombination mediator complex in genome maintenance and tumor suppression. Our findings may provide a paradigm for understanding the roles of DSS1 in other biological processes.
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•DSS1 works in conjunction with BRCA2 to facilitate RPA-RAD51 exchange on ssDNA•DSS1 interacts with RPA via its solvent-exposed acidic loop•DSS1 binds the RPA70 subunit of RPA and attenuates ssDNA binding by it•DSS1 could also function as a nucleic acid mimic in other biological processes
How RPA is displaced from DNA to enable RAD51 recombinase loading in homologous recombination (HR) is not known. Zhao et al. show that DSS1 targets RPA and mimics DNA to facilitate RPA displacement. This identifies DSS1 as an essential component of a HR mediator complex with the tumor suppressor BRCA2.
The tumor suppressor BRCA2 participates in DNA double-strand break repair by RAD51-dependent homologous recombination and protects stressed DNA replication forks from nucleolytic attack. We ...demonstrate that the C-terminal Recombinase Binding (CTRB) region of BRCA2, encoded by gene exon 27, harbors a DNA binding activity. CTRB alone stimulates the DNA strand exchange activity of RAD51 and permits the utilization of RPA-coated ssDNA by RAD51 for strand exchange. Moreover, CTRB functionally synergizes with the Oligonucleotide Binding fold containing DNA binding domain and BRC4 repeat of BRCA2 in RPA-RAD51 exchange on ssDNA. Importantly, we show that the DNA binding and RAD51 interaction attributes of the CTRB are crucial for homologous recombination and protection of replication forks against MRE11-mediated attrition. Our findings shed light on the role of the CTRB region in genome repair, reveal remarkable functional plasticity of BRCA2, and help explain why deletion of Brca2 exon 27 impacts upon embryonic lethality.
NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 ...shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.
The UAF1-USP1 complex deubiquitinates FANCD2 during execution of the Fanconi anemia DNA damage response pathway. As such, UAF1 depletion results in persistent FANCD2 ubiquitination and DNA damage ...hypersensitivity. UAF1-deficient cells are also impaired for DNA repair by homologous recombination. Herein, we show that UAF1 binds DNA and forms a dimeric complex with RAD51AP1, an accessory factor of the RAD51 recombinase, and a trimeric complex with RAD51 through RAD51AP1. Two small ubiquitin-like modifier (SUMO)-like domains in UAF1 and a SUMO-interacting motif in RAD51AP1 mediate complex formation. Importantly, UAF1 enhances RAD51-mediated homologous DNA pairing in a manner that is dependent on complex formation with RAD51AP1 but independent of USP1. Mechanistically, RAD51AP1-UAF1 co-operates with RAD51 to assemble the synaptic complex, a critical nucleoprotein intermediate in homologous recombination, and cellular studies reveal the biological significance of the RAD51AP1-UAF1 protein complex. Our findings provide insights into an apparently USP1-independent role of UAF1 in genome maintenance.
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•UAF1 possesses DNA binding activity•UAF1 synergizes with RAD51AP1 in the RAD51-mediated D-loop reaction•RAD51AP1-UAF1 promotes synaptic complex formation with the RAD51 recombinase•The RAD51AP1-UAF1 complex is indispensable for homologous recombination
Liang et al. find that UAF1 has DNA binding activity, define the interaction interface within the RAD51AP1-UAF1 complex, and show that the protein complex works in conjunction with the RAD51 presynaptic filament to mediate DNA pairing in homologous recombination.
Homologous recombination mediated by RAD51 recombinase helps eliminate chromosomal lesions, such as DNA double-strand breaks induced by radiation or arising from injured DNA replication forks. The ...tumor suppressors BRCA2 and PALB2 act together to deliver RAD51 to chromosomal lesions to initiate repair. Here we document a new function of PALB2: to enhance RAD51's ability to form the D loop. We show that PALB2 binds DNA and physically interacts with RAD51. Notably, although PALB2 alone stimulates D-loop formation, it has a cooperative effect with RAD51AP1, an enhancer of RAD51. This stimulation stems from the ability of PALB2 to function with RAD51 and RAD51AP1 to assemble the synaptic complex. Our results demonstrate the multifaceted role of PALB2 in chromosome damage repair. Because PALB2 mutations can cause cancer or Fanconi anemia, our findings shed light on the mechanism of tumor suppression in humans.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
To test the contribution of homologous recombinational repair (HRR) in repairing DNA damage sites induced by high-energy iron ions, we used (1) HRR-deficient rodent cells carrying a deletion in the ...RAD51D gene and (2) syngeneic human cells impaired for HRR by RAD51D or RAD51 knockdown using RNA interference. We found that in response to exposure to iron ions, HRR contributed to cell survival in rodent cells and that HRR deficiency abrogated RAD51 focus formation. Complementation of the HRR defect by human RAD51D rescues both enhanced cytotoxicity and RAD51 focus formation. For human cells irradiated with iron ions, cell survival was decreased, and in p53 mutant cells, the levels of mutagenesis were increased when HRR was impaired. Human cells synchronized in S phase exhibited a more pronounced resistance to iron ions compared with cells in G1 phase, and this increase in radioresistance was diminished by RAD51 knockdown. These results indicate a role for RAD51-mediated DNA repair (i.e. HRR) in removing a fraction of clustered lesions induced by charged-particle radiation. Our results are the first to directly show the requirement for an intact HRR pathway in human cells in ensuring DNA repair and cell survival after exposure to high-energy high-LET radiation.
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