GO:0010792 DNA double-strand break processing involved in repair via single-strand annealing: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0010792 describes the 5' to 3' exonucleolytic resection of DNA at a double-strand break (DSB) that generates a 3' single-strand DNA (ssDNA) overhang, enabling repair by single-strand annealing (SSA).
• SSA is a homology-directed repair pathway that anneals complementary ssDNA repeats flanking the break, typically resulting in deletion of the intervening sequence.
• The process is initiated by nucleases that resect the 5' strand, and in Saccharomyces cerevisiae this resection is promoted by Sae2 and Tel1.
• Ku70/Ku80 (HDF1/HDF2) influence the balance between non-homologous end joining and recombination, thereby affecting SSA usage.
• RAD51D and BRCA1 are homologous recombination factors that modulate repair fidelity and large deletions, with implications for genome stability.
• Experimental dissection of GO:0010792 requires combining resection assays, recombination reporters, and CRISPR-based genome editing to link mechanism to phenotype.
Description
GO:0010792, DNA double-strand break processing involved in repair via single-strand annealing, is a biological process that defines the early resection step committing a DSB to repair by single-strand annealing (SSA). In this pathway, the 5' ends at the break are exonucleolytically degraded in the 5' to 3' direction to expose 3' ssDNA overhangs that can anneal at flanking homologous repeats. This resection is a shared feature of homology-directed repair, but its coupling to SSA distinguishes it from other subpathways such as gene conversion or synthesis-dependent strand annealing. For researchers, GO:0010792 matters because SSA is inherently mutagenic: annealing between repeats that are not in perfect alignment leads to deletion of the intervening DNA, a hallmark of genome instability. The process is therefore central to understanding how cells balance faithful repair against mutagenic outcomes, and how defects in resection control contribute to disease. Mechanistically, SSA requires the coordinated action of nucleases and accessory factors that initiate resection, followed by strand annealing and flap removal. Genetic studies in yeast have shown that Sae2 and Tel1 promote ssDNA formation at breaks, while Ku70/Ku80 (HDF1/HDF2) modulate the choice between end joining and recombination. These findings provide a framework for interrogating the equivalent steps in human cells.
DNA double-strand break processing involved in repair via single-strand annealing At A Glance
| GO ID | GO:0010792 |
|---|---|
| GO term | DNA double-strand break processing involved in repair via single-strand annealing |
| Ontology | biological_process |
| Synonym | None |
| Major function | 5' to 3' exonucleolytic resection of DSB ends to generate 3' ssDNA overhangs for SSA |
| Pathway context | Homology-directed repair; single-strand annealing subpathway |
| Key outcome | Annealing of flanking repeats and deletion of intervening DNA |
| Associated factors | Sae2, Tel1, Ku70/Ku80, RAD51D, BRCA1 |
What Is GO:0010792?
GO:0010792 is defined as the 5' to 3' exonucleolytic resection of DNA at the site of a double-strand break to form a 3' single-strand DNA overhang that results in repair of the break via single-strand annealing. In other words, it is the processing step that creates the ssDNA substrate required for SSA, a homology-directed repair mechanism that anneals complementary sequences flanking the break.
Why Is DNA double-strand break processing involved in repair via single-strand annealing Important in Cell Biology?
GO:0010792 is important because it defines the commitment step to a mutagenic repair pathway that can cause deletions and genome rearrangements. Understanding this process helps explain how cells maintain genome stability and how defects in resection control contribute to cancer and other diseases. It also provides a mechanistic basis for interpreting CRISPR editing outcomes, where SSA-like repair can generate predictable deletions between repeats.
• SSA is a homology-directed repair pathway that can cause deletions between repeats, directly impacting genome stability.
• Resection at DSBs is a shared early step in homology-directed repair, making GO:0010792 central to repair pathway choice.
• Sae2 and Tel1 promote ssDNA formation at breaks in yeast, linking resection to checkpoint signaling.
• Ku70/Ku80 (HDF1/HDF2) influence recombination frequency, showing crosstalk between end joining and SSA.
• RAD51D protects the genome from large deletions, implicating SSA-like events in genomic instability.
• BRCA1 levels, controlled by NUSAP1, affect the DNA damage response and repair fidelity.
• Defects in resection and SSA are relevant to cancer predisposition and therapy resistance.
• CRISPR-based models can be used to dissect the genetic requirements for GO:0010792.
What Happens During DNA double-strand break processing involved in repair via single-strand annealing?
Initiation of 5' to 3' resection at the DSB
In simple terms: The broken DNA ends are chewed back to create single-stranded tails.
The process begins with nucleolytic degradation of the 5' strand at the DSB, generating 3' ssDNA overhangs. In Saccharomyces cerevisiae, this resection is promoted by Sae2 and Tel1, which are required for single-strand DNA formation at the break. This step is a prerequisite for SSA and other homology-directed repair subpathways.
Exonucleolytic processing and overhang formation
In simple terms: Enzymes continue to resect the ends, making longer single-stranded regions.
The 5' to 3' exonucleolytic resection extends the ssDNA overhangs, creating a substrate for homology search and annealing. The extent of resection influences which repair pathway is used, with SSA requiring sufficient ssDNA to anneal at flanking repeats. Ku70/Ku80 (HDF1/HDF2) modulate this step by competing with recombination factors, thereby affecting the balance between end joining and SSA.
Annealing of flanking repeats
In simple terms: The single-stranded tails find matching sequences and stick together.
Once 3' ssDNA overhangs are exposed, they can anneal at complementary sequences flanking the break, a defining feature of SSA. This annealing is mediated by DNA-pairing and annealing proteins that facilitate homology recognition. The result is a paired intermediate that often contains non-homologous flaps and intervening sequences.
Flap removal and ligation
In simple terms: Extra DNA tails are clipped and the break is sealed.
Following annealing, non-homologous 3' flaps are removed by nucleases, and the remaining nicks are ligated to restore an intact duplex. This step completes repair but frequently results in deletion of the DNA between the repeats. The overall outcome is a conservative repair event that can nonetheless be mutagenic.
Regulation by homologous recombination factors
In simple terms: Other repair proteins control whether SSA happens and how accurate it is.
RAD51D and BRCA1 are homologous recombination factors that influence repair fidelity and large deletion formation. NUSAP1 controls BRCA1 protein levels, thereby affecting the DNA damage response and potentially SSA usage. These regulatory inputs ensure that resection and annealing are coordinated with cell cycle and checkpoint signaling.
Key Genes Involved in GO:0010792 DNA double-strand break processing involved in repair via single-strand annealing
The following genes and proteins have been implicated in DSB processing and single-strand annealing, based on the verified literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| Sae2 | Promotes single-strand DNA formation at DSBs in yeast | Model for resection initiation and SSA commitment |
| Tel1 | ATM-like kinase required for ssDNA formation at breaks | Links checkpoint signaling to resection |
| HDF1 (Ku70) | Modulates recombination and end joining | Determines repair pathway choice |
| HDF2 (Ku80) | Modulates recombination and end joining | Determines repair pathway choice |
| RAD51D | Protects genome from large deletions | Homologous recombination fidelity |
| BRCA1 | DNA damage response and repair | Regulated by NUSAP1; affects repair |
| NUSAP1 | Controls BRCA1 protein levels | Upstream regulator of repair |
| RAD52 | DNA annealing in homologous recombination | Central to SSA annealing step |
| RPA | Binds ssDNA during resection | ssDNA protection and signaling |
| MRE11 | Part of resection machinery | Initiates 5' to 3' resection |
| RAD50 | Part of MRN complex | Resection and checkpoint activation |
| NBS1 | Part of MRN complex | Resection and damage signaling |
| EXO1 | Exonuclease that extends resection | Long-range resection |
| DMC1 | Meiotic recombinase | Homology search and strand exchange |
| RAD51 | Recombinase in homology-directed repair | Strand invasion and repair |
| BLM | Helicase that processes recombination intermediates | Regulates SSA and crossovers |
| GEN1 | Holliday junction resolvase | Resolution of recombination intermediates |
How Is DNA double-strand break processing involved in repair via single-strand annealing Regulated?
Regulation of GO:0010792 occurs at multiple levels. In yeast, Sae2 and Tel1 are required for single-strand DNA formation at breaks, linking resection to checkpoint signaling. Ku70/Ku80 (HDF1/HDF2) modulate the frequency of recombination, thereby influencing the choice between end joining and SSA. In human cells, NUSAP1 controls BRCA1 protein levels, which in turn affects the DNA damage response and repair fidelity. RAD51D also influences the formation of large deletions, suggesting that homologous recombination factors can suppress or promote SSA-like outcomes. These regulatory layers ensure that resection and annealing are coordinated with cell cycle progression and genome stability surveillance.
DNA double-strand break processing involved in repair via single-strand annealing and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| RAD51D | Homologous recombination deficiency, cancer | Knockout cell lines and organoids |
| BRCA1 | Breast/ovarian cancer, DNA damage response | Point mutation knock-in models |
| NUSAP1 | Regulates BRCA1; cancer | Overexpression and knockout models |
| HDF1 (Ku70) | Genome instability | Yeast deletion mutants |
| HDF2 (Ku80) | Genome instability | Yeast deletion mutants |
Cancer and genome instability
Defects in DSB processing and SSA can lead to large deletions and genome rearrangements, which are hallmarks of cancer. RAD51D protects the genome from large deletions, and its loss is associated with homologous recombination deficiency. BRCA1, regulated by NUSAP1, is a key tumor suppressor in breast and ovarian cancer, and its dysfunction impairs the DNA damage response.
Therapeutic implications
Tumors with defects in homology-directed repair, including SSA-related factors, may be sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors and other DNA-damaging agents. Understanding GO:0010792 helps identify which patients may benefit from these therapies.
Neurodegeneration and aging
Persistent DSBs and defective repair contribute to neuronal loss and aging. Although direct links between GO:0010792 and neurodegeneration are less established, the pathway's role in maintaining genome integrity suggests relevance to age-related diseases.
From DNA double-strand break processing involved in repair via single-strand annealing-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does gene X promote 5' to 3' resection? | Knockout cell lines with resection assays |
| Does a point mutation in gene X affect SSA? | Point mutation knock-in via CRISPR |
| Does gene X interact with Sae2/Tel1? | Tagged knock-in for co-IP |
| Does overexpression of gene X increase SSA? | Overexpression cell models |
| Does gene X loss cause large deletions? | Knockout and reporter assays |
| Does gene X regulate BRCA1 levels? | Knockout/overexpression with western blot |
How to Study the DNA double-strand break processing involved in repair via single-strand annealing Process
| Method | What It Measures | Typical Application |
|---|---|---|
| qPCR-based resection assay | ssDNA formation at DSB | Quantify 5' to 3' resection |
| SSA reporter | Frequency of annealing between repeats | Measure SSA efficiency |
| CRISPR-Cas9 editing | Repair outcomes at defined breaks | Test gene requirements |
| Co-immunoprecipitation | Protein-protein interactions | Identify resection complex components |
| Live-cell imaging | Recruitment kinetics of repair factors | Visualize resection foci |
| Western blot | Protein levels (e.g., BRCA1) | Assess regulation by NUSAP1 |
| Yeast genetics | Recombination frequency | Study Ku70/Ku80 roles |
| Sequencing of repair junctions | Deletion size and breakpoint | Characterize SSA products |
Resection assays
Resection can be measured by quantitative PCR or sequencing of ssDNA at defined DSBs. These assays detect the 5' to 3' degradation that defines GO:0010792.
SSA reporter systems
Direct repeat reporters flanking a nuclease site allow quantification of SSA frequency and deletion size. Such systems have been used to show conservative repair by two steps of annealing.
CRISPR-based genome editing
CRISPR-Cas9 can introduce DSBs at defined loci, and repair outcomes can be analyzed by sequencing to infer SSA usage. This approach enables structure-function studies of resection factors.
Protein interaction and localization
Co-immunoprecipitation, proximity labeling, and live-cell imaging can reveal how resection factors assemble at breaks. Tagged knock-in lines facilitate these studies.
How CRISPR Can Be Used to Study GO:0010792 DNA double-strand break processing involved in repair via single-strand annealing
Knockout
CRISPR knockout of candidate genes (e.g., RAD51D, BRCA1) can be used to test their requirement for DSB processing and SSA. Loss-of-function models reveal whether the gene is essential for 5' to 3' resection.
Point Mutation
Point mutations in resection factors can be introduced to dissect catalytic versus regulatory functions. Such models help determine which residues are critical for SSA.
Knock-in
Tagged knock-in of repair proteins enables visualization and interaction studies at endogenous levels. This is useful for tracking resection complex assembly.
Overexpression
Overexpression of genes such as NUSAP1 or BRCA1 can test whether increased dosage alters SSA and genome stability. These models complement knockout studies.
How EDITGENE Supports DNA double-strand break processing involved in repair via single-strand annealing Research
Researchers studying DNA double-strand break processing involved in repair via single-strand annealing-related genes often need to determine whether a candidate gene is causally involved in resection, annealing, or repair fidelity. EDITGENE provides the tools to build such causal models.
Contact EDITGENE today to design your custom CRISPR model for DNA double-strand break processing involved in repair via single-strand annealing research.
Frequently Asked Questions About DNA double-strand break processing involved in repair via single-strand annealing
What is GO:0010792?
GO:0010792 is the biological process of 5' to 3' exonucleolytic resection at a DNA double-strand break to form 3' single-strand DNA overhangs that enable repair via single-strand annealing.
What genes are involved in DNA double-strand break processing involved in repair via single-strand annealing?
Key genes include Sae2, Tel1, HDF1 (Ku70), HDF2 (Ku80), RAD51D, BRCA1, and NUSAP1, among others.
How does single-strand annealing repair a double-strand break?
It anneals complementary single-stranded repeats flanking the break, leading to deletion of the intervening DNA.
What is the difference between SSA and homologous recombination?
SSA is a homology-directed repair subpathway that anneals repeats and causes deletions, whereas other HR subpathways may use a template for accurate repair.
Which proteins promote 5' to 3' resection at DSBs?
Sae2 and Tel1 promote single-strand DNA formation in yeast, and the MRN complex with EXO1 mediates resection in human cells.
How is SSA regulated?
It is regulated by checkpoint kinases, Ku70/Ku80, and homologous recombination factors such as BRCA1 and RAD51D.
What diseases are linked to defective DSB processing?
Defects are linked to cancer predisposition and genome instability, particularly in homologous recombination-deficient tumors.
How can I study GO:0010792 in the lab?
Use resection assays, SSA reporters, and CRISPR-based editing to measure repair outcomes.
What is the role of RAD51D in SSA?
RAD51D protects the genome from large deletions, suggesting it suppresses mutagenic SSA-like events.
Can CRISPR be used to model SSA defects?
Yes, CRISPR knockout or point mutation of resection genes can model SSA defects and reveal their impact on genome stability.
Conclusion
GO:0010792 defines the resection step that commits a DSB to repair by single-strand annealing, a pathway that is both conservative and mutagenic. Its regulation by Sae2, Tel1, Ku70/Ku80, and homologous recombination factors such as RAD51D and BRCA1 underscores its importance in genome stability and disease. By combining CRISPR-based models with resection and SSA assays, researchers can dissect the genetic requirements for this process and identify therapeutic vulnerabilities.
References
- 1. Morrical SW. 2015. DNA-pairing and annealing processes in homologous recombination and homology-directed repair.. Cold Spring Harb Perspect Biol 7(2):a016444 PMID: 25646379
- 2. Storici F et al.. 2006. Conservative repair of a chromosomal double-strand break by single-strand DNA through two steps of annealing.. Mol Cell Biol 26(20):7645-57 PMID: 16908537
- 3. Reh WA et al.. 2017. The homologous recombination protein RAD51D protects the genome from large deletions.. Nucleic Acids Res 45(4):1835-1847 PMID: 27924006
- 4. Kotian S et al.. 2014. NUSAP1 influences the DNA damage response by controlling BRCA1 protein levels.. Cancer Biol Ther 15(5):533-43 PMID: 24521615
- 5. Lee K et al.. 2007. Saccharomyces cerevisiae Sae2- and Tel1-dependent single-strand DNA formation at DNA break promotes microhomology-mediated end joining.. Genetics 176(4):2003-14 PMID: 17565964
- 6. Cervelli T et al.. 2000. Effects of HDF1 (Ku70) and HDF2 (Ku80) on spontaneous and DNA damage-induced intrachromosomal recombination in Saccharomyces cerevisiae.. Mol Gen Genet 264(1-2):56-63 PMID: 11016833
