preparation of g1 synchronized cells to measure ssdna foci formation upon genotoxic stress

Detection of G1 Phase Single-Stranded DNA Foci via RPA2 Immunofluorescent Staining

To sustain life, cells constantly survey and repair DNA to maintain their genomic integrity. Cells may accumulate various types of DNA damage due to both endogenous (e.g., oxidation, alkylation, deamination, replication errors) and exogenous (e.g., UV, ionizing irradiation) sources of DNA stressors. Failure to repair these lesions results in either apoptosis, cell cycle arrest, or senescence and can lead to diseases1. DNA lesions could be addressed by any of the following main DNA repair pathways: DR (direct reversal repair), which mainly repairs alkylated bases2; BER (base excision repair), which targets non-bulky DNA bases errors and single-stranded DNA breaks (SSBs)3; NER (nucleotide excision repair) correcting bulky, helix-distorting DNA lesions4; MMR (mismatch repair) mainly targeting DNA mismatches, insertion/deletion loops (IDLs), and certain base damages5; NHEJ (non-homologous end joining) and HRR (homologous recombination repair) that are both active at double-stranded DNA breaks (DSBs)6; and TLS (translesion synthesis), which is a DNA lesion bypass mechanism7. Though these pathways have distinct substrate specificities, there are certain overlaps amongst them to ensure redundancy for efficient repair. Understanding the action of the different DNA repair pathways in various cell cycle phases is crucial as these DNA repair factors could serve as essential targets for therapeutic approaches to treat cancer, aging, and neurological disorders8,9.
Single-stranded DNA (ssDNA) is generated throughout the cell cycle due to the repair of DNA lesions generated by both endogenous and exogenous DNA-damaging agents. Upon genotoxic stress, ssDNA is generated abundantly in the S and G2 phases where HRR and MMR have their highest activity and when the replication machinery stalls or collapses when encountering DNA lesions6,10,11. Other DNA repair pathways (e.g., NHEJ/microhomology-mediated end joining (MMEJ)/single-strand annealing [SSA]) also generate ssDNA during DSB repair12. These ssDNA tracks usually arise from DNA resection, carried out by exonucleases such as EXO1, DNA2, and CtIP during HR and MMR, endonucleases such as XPF and XPG during NER, or through the combined action of POLB and FEN1 during BER4,13,14,15,16,17,18,19. Due to the work of the replication machinery, ssDNA tracks are also generated when the DNA helicases unwind DNA in front of the PCNA-bound replicative polymerases20. In contrast, in the G1 phase, the lack of HRR and DNA replication and the limited activity of MMR reduces the extent of ssDNA tracks generated and are therefore more challenging to detect10,11,21.
Cellular ssDNA tracks are highly sensitive structures that must be protected to avoid the formation of DSBs. This is achieved by coating the ssDNA tracks with RPA. RPA is an abundant heterotrimeric protein complex composed of multiple subunits (RPA1, RPA2, and RPA3, also referred to as RPA70, RPA32, and RPA14, respectively), which are ubiquitously expressed throughout the cell cycle22. Each RPA subunit contains a DNA-binding domain (DBD), capable of interacting with 4-6 nucleotides, and the combined subunits form a stable trimerization core. Altogether, RPA binds to approximately 20-30 nucleotides with sub-nanomolar affinity23,24.
Conventional methods use immunofluorescence (IF) microscopy to visualize ssDNA foci by labeling 5-bromo-2&#39;-deoxyuridine (BrdU) incorporated into genomic DNA using BrdU antibodies25. This approach relies on the fact that BrdU antibodies can only detect BrdU in exposed ssDNA25. Although this approach is straightforward, it also displays certain limitations. For instance, cells are pretreated to incorporate BrdU before the start of the experiment, which is time-consuming and can interfere with downstream effectors. Therefore, BrdU-based ssDNA detection is limited to replicating cells and cannot be used for quiescent cells. This excludes the application of this method to study DNA repair in non-replicating cells despite its importance in several diseases such as cancer and neurodegeneration5,26. Additionally, because the structures of BrdU and EdU are very similar, most BrdU antibodies display crossreactivity towards EdU, which must be considered when aiming for dual labeling experiments27. RPA staining has been previously utilized to show ssDNA foci mainly in S phase cells; however, some papers have also successfully used it outside the S phase28,29,30,31,32,33,34,35. The following protocol efficiently utilizes the properties of RPA, allowing the visualization of ssDNA foci following DNA damage in the G1 phase of the cell cycle (although it can be used in all cell cycle phases).

Author Spotlight: Visualizing Single-Stranded DNA During DNA Repair for Therapeutic Insights 

Visualizing Single-Stranded DNA Foci in the G1 Phase of the Cell Cycle

DNA damage response is indispensable for life. As DNA is constantly exposed to various damaging engines, lack of their repair leads to genomes'instability and diseases. We developed a method based on biological property of the RPA2, to visualize single strand DNA tract during DNA repair in different cell cycle stages.Traditional method for single strand DNA detection utilize BrdU antibodies. This approach can be used for actively replicating cells. Additionally, this antibody tend to cross-react with other nucleotide analogs, limiting their use.Unlike BRDU, our approach has a higher signal to noise ratio, making it usable in every cycle phase. Understanding the action of different DNA repair pathways in various cell cycle phases and potentially non replicating cells is crucial. Our protocol provides a new approach and tool to visualize single stranded DNA foci, where classical approaches utilizing BrdU are limited.A comprehensive understanding of DNA repair processes could serve as therapeutic targets to treat cancer, aging, and neurological diseases. Most of our cells are terminally differentiated and therefore do not replicate and divide. It is crucial to gain a better understanding of how these cells repair various DNA lesions.Therefore, our laboratory will be mainly focusing on post replicative DNA repair processes and developing new tools to study them.

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Preparation of G1 Synchronized Cells to Measure ssDNA Foci Formation upon Genotoxic Stress  

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333 Views.  NYU Grossman School of Medicine. Preparation of G1 Synchronized Cells to Measure ssDNA Foci Formation upon Genotoxic Stress

Video: Preparation of G1 Synchronized Cells to Measure ssDNA Foci Formation upon Genotoxic Stress  

DNA has dedicated cellular repair pathways capable of coping with lesions that could arise from both endogenous and/or exogenous sources. DNA repair necessitates collaboration between numerous proteins, responsible for covering a broad range of tasks from recognizing and signaling the presence of a DNA lesion to physically repairing it. During this process, tracks of single-stranded DNA (ssDNA) are often created, which are eventually filled by DNA polymerases. The nature of these ssDNA tracks (in terms of both length and number), along with the polymerase recruited to fill these gaps, are repair pathway-specific. The visualization of these ssDNA tracks can help us understand the complicated dynamics of DNA repair mechanisms.
This protocol provides a detailed method for the preparation of G1 synchronized cells to measure ssDNA foci formation upon genotoxic stress. Using an easy-to-utilize immunofluorescence approach, we visualize ssDNA by staining for RPA2, a component of the heterotrimeric replication protein A complex (RPA). RPA2 binds to and stabilizes ssDNA intermediates that arise upon genotoxic stress or replication to control DNA repair and DNA damage checkpoint activation. 5-Ethynyl-2'-deoxyuridine (EdU) staining is used to visualize DNA replication to exclude any S phase cells. This protocol provides an alternative approach to the conventional, non-denaturing 5-bromo-2'-deoxyuridine (BrdU)-based assays and is better suited for the detection of ssDNA foci outside the S phase.

The following protocol presents the detection of single-stranded DNA foci in the G1 phase of the cell cycle utilizing cell cycle synchronization followed by RPA2 immunofluorescent staining.

DNA has dedicated cellular repair pathways capable of coping with lesions that could arise from both endogenous and/or exogenous sources. DNA repair ...