DNA serves as the fundamental blueprint for all life. This intricate molecule, however, is constantly exposed to various damaging agents, which can compromise the genetic code, leading to cellular dysfunction or disease. To counteract these threats, cells possess an array of DNA repair pathways that restore the DNA’s original structure and maintain genomic stability. Single Strand Annealing (SSA) is one such pathway, addressing specific types of DNA damage.
The Basics of Single Strand Annealing
Single Strand Annealing (SSA) is a DNA repair mechanism primarily involved in fixing a severe form of DNA damage known as double-strand breaks (DSBs). These breaks involve both DNA strands being severed, posing a significant threat to genetic stability. SSA relies on the presence of homologous repetitive DNA sequences located on either side of the break site.
This pathway is considered a non-conservative repair mechanism because it leads to the loss of genetic material. When SSA is utilized, the DNA sequences between the two repetitive elements, as well as one of the repeats, are deleted. This outcome highlights its potential to induce genomic rearrangements and deletions. While it can be mutagenic, SSA is a conserved pathway, observed in various organisms, including mammalian cells, yeast, and plants.
How Single Strand Annealing Works
Single Strand Annealing initiates with the detection of a double-strand break. The broken DNA ends undergo resection, where exonuclease enzymes like EXO1, DNA2, or BLM helicase remove nucleotides from the 5′ ends, creating single-stranded DNA (ssDNA) overhangs with exposed 3′ ends. This continues until homologous repetitive sequences flanking the break are exposed on these single strands.
Once these complementary sequences are revealed, proteins like RAD52 facilitate their annealing. RAD52 is a DNA binding protein that mediates the annealing of ssDNA substrates, bringing the two homologous repeats together. This forms a synapsed intermediate, where the repetitive sequences align and base-pair.
After annealing, any non-homologous single-stranded DNA tails or “flaps” that protrude from the aligned region must be removed. The ERCC1/XPF protein complex, a nuclease, is responsible for cleaving these 3′ ssDNA tails. Finally, any remaining gaps are filled by DNA polymerases, and DNA ligase seals the nicks in the phosphodiester backbone, completing the repair process.
Significance of Single Strand Annealing
Single Strand Annealing plays a role in maintaining genomic stability, particularly in regions of the genome rich in repetitive DNA sequences. While it effectively repairs double-strand breaks, its mechanism of deletion can lead to significant genomic rearrangements. These deletions can alter gene dosage, disrupt gene function, or create fusion genes, potentially contributing to various genetic disorders.
The genomic alterations induced by SSA are also implicated in the development and progression of diseases such as cancer. The loss of tumor suppressor genes or the activation of oncogenes through SSA-mediated deletions can promote uncontrolled cell growth and proliferation. Understanding the conditions that favor SSA over other, more accurate repair pathways is an ongoing area of research.
In research and biotechnology, SSA has seen some application, though it is less commonly used than other gene-editing tools like CRISPR-Cas9. However, the principles of SSA, particularly the annealing of single-stranded DNA, have been leveraged in some gene-editing strategies, such as the development of RecT Editor via Designer-Cas9-Initiated Targeting (REDIT). This approach utilizes phage single-stranded DNA-annealing proteins to improve the efficiency of inserting kilobase-scale exogenous sequences into defined genomic regions, showing potential therapeutic application.