scholarly journals RAD-ical New Insights into RAD51 Regulation

2018 ◽  
Author(s):  
Meghan R. Sullivan ◽  
Kara A. Bernstein
Keyword(s):  
Genome Stability ◽  
Homology Search ◽  
Model Organisms ◽  
Filament Formation ◽  
Dna Double Strand Breaks ◽  
Current Perspective ◽  
Rad51 Paralogs ◽  
Human Rad51

Accurate repair of DNA is critical for genome stability and cancer prevention. DNA double-strand breaks are one of the most toxic lesions and can be repaired using homologous recombination (HR). HR is a high-fidelity DNA repair pathway that uses a homologous template for repair. One central HR step is RAD51 nucleoprotein filament formation on the single-stranded DNA ends, a step required for the homology search and strand invasion steps of HR. RAD51 filament formation is tightly controlled by many positive and negative regulators, collectively termed the RAD51 mediators. The RAD51 mediators function to nucleate, elongate, stabilize, and disassemble RAD51 during repair. In model organisms, RAD51 paralogs are RAD51 mediator proteins that structurally resemble RAD51 and promote its HR activity. New functions for the RAD51 paralogs during replication and in RAD51 filament flexibility have recently been uncovered. Mutations in the human RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and SWSAP1) are found in a subset of breast and ovarian cancers. Despite their discovery three decades ago, few advances have been made in understanding the function of the human RAD51 paralogs. Here we discuss the current perspective on the RAD51 paralogs in vivo and in vitro function and their relationship with cancer in vertebrate models.

scholarly journals RAD-ical New Insights into RAD51 Regulation

Genes ◽  
2018 ◽  
Vol 9 (12) ◽  
pp. 629 ◽  
Author(s):  
Meghan R. Sullivan ◽  
Kara A. Bernstein
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Homology Search ◽  
Model Organisms ◽  
Filament Formation ◽  
Dna Double Strand Breaks ◽  
Rad51 Paralogs ◽  
Human Rad51

The accurate repair of DNA is critical for genome stability and cancer prevention. DNA double-strand breaks are one of the most toxic lesions; however, they can be repaired using homologous recombination. Homologous recombination is a high-fidelity DNA repair pathway that uses a homologous template for repair. One central HR step is RAD51 nucleoprotein filament formation on the single-stranded DNA ends, which is a step required for the homology search and strand invasion steps of HR. RAD51 filament formation is tightly controlled by many positive and negative regulators, which are collectively termed the RAD51 mediators. The RAD51 mediators function to nucleate, elongate, stabilize, and disassemble RAD51 during repair. In model organisms, RAD51 paralogs are RAD51 mediator proteins that structurally resemble RAD51 and promote its HR activity. New functions for the RAD51 paralogs during replication and in RAD51 filament flexibility have recently been uncovered. Mutations in the human RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and SWSAP1) are found in a subset of breast and ovarian cancers. Despite their discovery three decades ago, few advances have been made in understanding the function of the human RAD51 paralogs. Here, we discuss the current perspective on the in vivo and in vitro function of the RAD51 paralogs, and their relationship with cancer in vertebrate models.

scholarly journals Rad52-Rad51 association is essential to protect Rad51 filaments against Srs2, but facultative for filament formation

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Emilie Ma ◽  
Pauline Dupaigne ◽  
Laurent Maloisel ◽  
Raphaël Guerois ◽  
Eric Le Cam ◽  
...  
Keyword(s):  
Current Model ◽  
Homology Search ◽  
Filament Formation ◽  
Dna Damaging Agents ◽  
Filament Assembly ◽  
Homologous Recombination Process ◽  
Key Steps

Homology search and strand exchange mediated by Rad51 nucleoprotein filaments are key steps of the homologous recombination process. In budding yeast, Rad52 is the main mediator of Rad51 filament formation, thereby playing an essential role. The current model assumes that Rad51 filament formation requires the interaction between Rad52 and Rad51. However, we report here that Rad52 mutations that disrupt this interaction do not affect γ-ray- or HO endonuclease-induced gene conversion frequencies. In vivo and in vitro studies confirmed that Rad51 filaments formation is not affected by these mutations. Instead, we found that Rad52-Rad51 association makes Rad51 filaments toxic in Srs2-deficient cells after exposure to DNA damaging agents, independently of Rad52 role in Rad51 filament assembly. Importantly, we also demonstrated that Rad52 is essential for protecting Rad51 filaments against dissociation by the Srs2 DNA translocase. Our findings open new perspectives in the understanding of the role of Rad52 in eukaryotes.

scholarly journals Rad52 oligomeric N-terminal domain stabilizes Rad51 nucleoprotein filaments and contributes to their protection against Srs2

2021 ◽  
Author(s):  
Emilie Ma ◽  
Laurent Maloisel ◽  
Lea Le Falher ◽  
Raphael Guerois ◽  
Eric Coic
Keyword(s):  
Genome Stability ◽  
Ring Structure ◽  
Homologous Sequence ◽  
Filament Formation ◽  
Repair Synthesis ◽  
Terminal Domain ◽  
Mediator Protein ◽  
Dna Repair Synthesis

Homologous recombination (HR) depends on the formation of a nucleoprotein filament of the recombinase Rad51 to scan the genome and invade the homologous sequence used as template for DNA repair synthesis. Therefore, HR is highly accurate and crucial for genome stability. Rad51 filament formation is controlled by positive and negative factors. In Saccharomyces cerevisiae, the mediator protein Rad52 catalyzes Rad51 filament formation and stabilizes them, mostly by counteracting the disruptive activity of the translocase Srs2. Srs2 activity is essential to avoid the formation of toxic Rad51 filaments, as revealed by Srs2-deficient cells. We previously reported that Rad52 SUMOylation or mutations disrupting the Rad52-Rad51 interaction suppress Rad51 filament toxicity because they disengage Rad52 from Rad51 filaments and reduce their stability. Here, we found that mutations in Rad52 N-terminal domain also suppress the DNA damage sensitivity of Srs2-deficient cells without disturbing Rad52 mediator and pairing activity, both in vivo and in vitro. Structural studies showed that these mutations affect the Rad52 oligomeric ring structure. Overall, our findings indicate that Rad52 ring structure is important for protecting Rad51 filaments from Srs2, but can increase Rad51 filament stability and toxicity in Srs2-deficient cells. This stabilization function is distinct from Rad52 mediator and annealing activities.

scholarly journals POGZ modulates the DNA damage response in a HP1-dependent manner

2021 ◽  
Author(s):  
John Heath ◽  
Estelle Simo Cheyou ◽  
Steven Findlay ◽  
Vincent Luo ◽  
Edgar Pinedo Carpio ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Genome Stability ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Humoral Immune ◽  
Damage Response

The heterochromatin protein HP1 plays a central role in the maintenance of genome stability, in particular by promoting homologous recombination (HR)-mediated DNA repair. However, little is still known about how HP1 is controlled during this process. Here, we describe a novel function of the POGO transposable element derived with ZNF domain protein (POGZ) in the regulation of HP1 during the DNA damage response in vitro. POGZ depletion delays the resolution of DNA double-strand breaks (DSBs) and correlates with an increased sensitivity to different DNA damaging agents, including the clinically-relevant Cisplatin and Talazoparib. Mechanistically, POGZ promotes homology-directed DNA repair pathways by retaining the BRCA1/BARD1 complex at DSBs, in a HP1-dependent manner. In vivo CRISPR inactivation of Pogz is embryonic lethal and Pogz haplo-insufficiency (Pogz+/Δ) results in a developmental delay, a deficit in intellectual abilities, a hyperactive behaviour as well as a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Importantly, Pogz+/Δ mice are radiosensitive and accumulate DSBs in diverse tissues, including the spleen and the brain. Altogether, our findings identify POGZ as an important player in homology-directed DNA repair both in vitro and in vivo, with clinical implications for the WHSUS.

scholarly journals Rad52 Oligomeric N-Terminal Domain Stabilizes Rad51 Nucleoprotein Filaments and Contributes to Their Protection against Srs2

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1467
Author(s):  
Emilie Ma ◽  
Laurent Maloisel ◽  
Léa Le Falher ◽  
Raphaël Guérois ◽  
Eric Coïc
Keyword(s):  
Genome Stability ◽  
Ring Structure ◽  
Homologous Sequence ◽  
Filament Formation ◽  
Repair Synthesis ◽  
Terminal Domain ◽  
Mediator Protein ◽  
Dna Repair Synthesis

Homologous recombination (HR) depends on the formation of a nucleoprotein filament of the recombinase Rad51 to scan the genome and invade the homologous sequence used as a template for DNA repair synthesis. Therefore, HR is highly accurate and crucial for genome stability. Rad51 filament formation is controlled by positive and negative factors. In Saccharomyces cerevisiae, the mediator protein Rad52 catalyzes Rad51 filament formation and stabilizes them, mostly by counteracting the disruptive activity of the translocase Srs2. Srs2 activity is essential to avoid the formation of toxic Rad51 filaments, as revealed by Srs2-deficient cells. We previously reported that Rad52 SUMOylation or mutations disrupting the Rad52–Rad51 interaction suppress Rad51 filament toxicity because they disengage Rad52 from Rad51 filaments and reduce their stability. Here, we found that mutations in Rad52 N-terminal domain also suppress the DNA damage sensitivity of Srs2-deficient cells. Structural studies showed that these mutations affect the Rad52 oligomeric ring structure. Overall, in vivo and in vitro analyzes of these mutants indicate that Rad52 ring structure is important for protecting Rad51 filaments from Srs2, but can increase Rad51 filament stability and toxicity in Srs2-deficient cells. This stabilization function is distinct from Rad52 mediator and annealing activities.

scholarly journals The Yeast Recombinational Repair Protein Rad59 Interacts With Rad52 and Stimulates Single-Strand Annealing

Genetics ◽  
2001 ◽  
Vol 159 (2) ◽  
pp. 515-525 ◽  
Author(s):  
Allison P Davis ◽  
Lorraine S Symington
Keyword(s):  
Protein A ◽  
Single Strand ◽  
Physical Interaction ◽  
Dna Double Strand Breaks ◽  
Single Stranded Dna ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Recombination Pathway

Abstract The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.

Computational study for suppression of CD25/IL-2 interaction

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Moein Dehbashi ◽  
Zohreh Hojati ◽  
Majid Motovali-bashi ◽  
Mazdak Ganjalikhani-Hakemi ◽  
Akihiro Shimosaka ◽  
...  
Keyword(s):  
Small Molecules ◽  
Cancer Progression ◽  
Immune Escape ◽  
De Novo ◽  
Computational Study ◽  
Treg Cells ◽  
Homology Search ◽  
Small Interfering Rnas

AbstractCancer recurrence presents a huge challenge in cancer patient management. Immune escape is a key mechanism of cancer progression and metastatic dissemination. CD25 is expressed in regulatory T (Treg) cells including tumor-infiltrating Treg cells (TI-Tregs). These cells specially activate and reinforce immune escape mechanism of cancers. The suppression of CD25/IL-2 interaction would be useful against Treg cells activation and ultimately immune escape of cancer. Here, software, web servers and databases were used, at which in silico designed small interfering RNAs (siRNAs), de novo designed peptides and virtual screened small molecules against CD25 were introduced for the prospect of eliminating cancer immune escape and obtaining successful treatment. We obtained siRNAs with low off-target effects. Further, small molecules based on the binding homology search in ligand and receptor similarity were introduced. Finally, the critical amino acids on CD25 were targeted by a de novo designed peptide with disulfide bond. Hence we introduced computational-based antagonists to lay a foundation for further in vitro and in vivo studies.

scholarly journals Vimentin provides the mechanical resilience required for amoeboid migration and protection of the nucleus

2019 ◽  
Author(s):  
Luiza Da Cunha Stankevicins ◽  
Marta Urbanska ◽  
Daniel AD. Flormann ◽  
Emmanuel Terriac ◽  
Zahra Mostajeran ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dendritic Cells ◽  
Cell Migration ◽  
Molecular Mechanisms ◽  
Cell Cortex ◽  
Dna Double Strand Breaks ◽  
Nuclear Positioning ◽  
Mechanical Stiffness

AbstractDendritic cells use amoeboid migration through constricted passages to reach the lymph nodes, and this homing function is crucial for immune responses. Amoeboid migration requires mechanical resilience, however, the underlying molecular mechanisms for this type of migration remain unknown. Because vimentin intermediate filaments (IFs) and microfilaments regulate adhesion-dependent migration in a bidirectional manner, we analyzed if they exert a similar control on amoeboid migration. Vimentin was required for cellular resilience, via a joint interaction between vimentin IFs and F-actin. Reduced actin mobility in the cell cortex of vimentin-reduced cells indicated that vimentin promotes Factin subunit exchange and dynamics. These mechano-dynamic alterations in vimentin-deficient dendritic cells impaired amoeboid migration in confined environments in vitro and blocked lymph node homing in mouse experiments in vivo. Correct nuclear positioning is important in confined amoeboid migration both to minimize resistance and to avoid DNA damage. Vimentin-deficiency also led to DNA double strand breaks in the compressed dendritic cells, pointing to a role of vimentin in nuclear positioning. Together, these observations show that vimentin IF-microfilament interactions provide both the specific mechano-dynamics required for dendritic cell migration and the protection the genome needs in compressed spaces.Summary statementVimentin — in joint action with actin — mediates the mechanical stiffness of cells required for amoeboid cell migration through confined spaces and protects the nucleus from DNA damage.

scholarly journals MCM7 Ubiquitination Regulates CMG Helicase Disassembly in Human

2021 ◽  
Author(s):  
Qianqian Sun ◽  
Kun Liu ◽  
Fangzhou Li ◽  
Bingquan Qiu ◽  
Zhisong Fu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Genome Stability ◽  
Human Tumor ◽  
Cell Fractionation ◽  
Minichromosome Maintenance ◽  
E3 Ligases ◽  
Minichromosome Maintenance Protein ◽  
Fork Stalling

Abstract BackgroundThe disassembly of the replisome plays an essential role in maintaining genome stability at the termination of DNA replication. However, the mechanism of replisome disassembly remains unknown in human. In this study, we screened E3 ligases and deubiquitinases (DUBs) for the ubiquitination of minichromosome maintenance protein (MCM) 7 and provided evidence of this process driving CMG helicase disassembly in human tumor cells. MethodsSILAC-MS/MS was analyzed to identify ubiquitinated proteins in HeLa cells. The ubiquitination/deubiquitylation assay in vitro and in vivo were detected by Western blot. Thymidine and HU were implied to synchronized cell cycle，and detect the role of ubiquitinated MCM7 in cell cycle. Cell fractionation assay was used to detect the function of ubiquitination of MCM7 in chromatin and non-chromatin. Aphidicolin、Etoposide、ICRF-193 and IR were applied to cause replication fork stalling. MG-132 and NMS-873 were used to inhibit the proteasome degradation and p97 segregase. Flow cytometer and FlowJo flow cytometry software were used to cell cycle analysis.ResultsIn our study, we found that the ubiquitin ligase RNF8 catalyzes the k63-linked poly-ubiquitination of MCM7 both in vivo and in vitro, and lysine 145 of MCM7 is the primary ubiquitination site. Moreover, the poly-ubiquitination of MCM7 mainly exists in the chromatin, which is dynamically regulated by the cell cycle, mainly occurs in the late S phase. And DNA damage can significantly reduce the poly-ubiquitylation of MCM7 in the late S phage. Furthermore, the proteasome, p97 segregase, USP29 and ATXN3 are required for the removal of MCM7 ubiquitination to promote the disassembly of CMG on chromatin. ConclusionsIn the late S phage of cell cycle, RNF8 catalyzes the poly-ubiquitination of MCM7, and then initiates the disassembly of CMG helicase from chromatin, which is mediated by p97, proteasome, USP29 and ATXN3 in human. We reveal the novel function of the poly-ubiquitylation of MCM7, which is a regulatory signal to control CMG complex unloading at replication termination sites.

scholarly journals DNA double-strand break repair: a theoretical framework and its application

2016 ◽  
Vol 13 (114) ◽  
pp. 20150679 ◽  
Author(s):  
Philip J. Murray ◽  
Bart Cornelissen ◽  
Katherine A. Vallis ◽  
S. Jon Chapman
Keyword(s):  
Dna Damage ◽  
Auger Electron ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Histone H2ax ◽  
Dna Double Strand Break ◽  
A Cell

DNA double-strand breaks (DSBs) are formed as a result of genotoxic insults, such as exogenous ionizing radiation, and are among the most serious types of DNA damage. One of the earliest molecular responses following DSB formation is the phosphorylation of the histone H2AX, giving rise to γ H2AX. Many copies of γ H2AX are generated at DSBs and can be detected in vitro as foci using well-established immuno-histochemical methods. It has previously been shown that anti- γ H2AX antibodies, modified by the addition of the cell-penetrating peptide TAT and a fluorescent or radionuclide label, can be used to visualize and quantify DSBs in vivo . Moreover, when labelled with a high amount of the short-range, Auger electron-emitting radioisotope, 111 In, the amount of DNA damage within a cell can be increased, leading to cell death. In this report, we develop a mathematical model that describes how molecular processes at individual sites of DNA damage give rise to quantifiable foci. Equations that describe stochastic mean behaviours at individual DSB sites are derived and parametrized using population-scale, time-series measurements from two different cancer cell lines. The model is used to examine two case studies in which the introduction of an antibody (anti- γ H2AX-TAT) that targets a key component in the DSB repair pathway influences system behaviour. We investigate: (i) how the interaction between anti- γ H2AX-TAT and γ H2AX effects the kinetics of H2AX phosphorylation and DSB repair and (ii) model behaviour when the anti- γ H2AX antibody is labelled with Auger electron-emitting 111 In and can thus instigate additional DNA damage. This work supports the conclusion that DSB kinetics are largely unaffected by the introduction of the anti- γ H2AX antibody, a result that has been validated experimentally, and hence the hypothesis that the use of anti- γ H2AX antibody to quantify DSBs does not violate the image tracer principle. Moreover, it provides a novel model of DNA damage accumulation in the presence of Auger electron-emitting 111 In that is supported qualitatively by the available experimental data.

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