DNA is constantly under threat from internal and external factors that can lead to damage. The most severe form is a Double-Strand Break (DSB), where both strands of the DNA helix are severed simultaneously. If left unrepaired, a single DSB can lead to massive loss of genetic information, chromosomal aberrations, or cell death. Cells have evolved intricate repair mechanisms, including Alternative End Joining (A-EJ), also known as Microhomology-Mediated End Joining (MMEJ). This pathway is an error-prone strategy for re-ligating the broken ends of the DNA molecule.
The Molecular Steps of Alternative End Joining
The process begins when sensing proteins, such as Poly(ADP-ribose) polymerase 1 (PARP-1), rapidly bind to the DSB site. PARP-1 mediates the recruitment of the Mre11-Rad50-Nbs1 (MRN) complex, which processes the broken DNA ends. This action triggers DNA end resection, the trimming back of the 5′ DNA strand at the break site.
This resection is extensive, involving the MRN complex, CtIP, and nucleases like Exo1 or DNA2. The goal is to generate long, single-stranded DNA (ssDNA) overhangs that expose short, repetitive sequences known as microhomologies. These microhomologies are typically two to 20 nucleotides in length and exist on either side of the break.
The ssDNA ends align based on these exposed microhomologies, juxtaposing the broken ends. Once paired, the intervening DNA sequences—the stretch of DNA between the two aligned homologies—are excised by nucleases. This excision causes the loss of genetic material, resulting in a characteristic deletion at the repair site.
The remaining gaps are filled by specialized DNA polymerases, such as Polymerase theta (PolQ), which bridges the gaps using the paired microhomologies as a primer. PolQ has low fidelity, which can introduce sequence aberrations, including small insertions, during synthesis. The final step involves sealing the remaining nicks by DNA Ligase 3 (LIG3), completing the rejoining of the two broken DNA strands.
A-EJ as a Backup System for DNA Repair
The cell possesses two primary, highly efficient pathways for fixing double-strand breaks: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). NHEJ is a fast, dominant repair mechanism operating throughout all phases of the cell cycle, especially in the G1 phase when a sister chromatid template is unavailable. HR is a high-fidelity, template-dependent pathway restricted to the S and G2 phases when an identical sister chromatid is present to serve as a repair template.
A-EJ is considered an “alternative” because it acts as a secondary or backup mechanism, typically becoming active when these primary pathways are compromised. If core components of NHEJ, such as the Ku protein, are absent or defective, the cell switches to A-EJ to complete the repair. This switch ensures cell survival by providing a last resort for dealing with DSBs.
The choice of repair pathway is a complex decision, but A-EJ is often forced when the HR pathway is unavailable or defective, such as in cancer cells with mutations in the BRCA genes. Compared to the fast joining of NHEJ or the accurate repair of HR, A-EJ is a slower process that relies on extensive DNA end resection to find microhomologies. This reliance on short, repetitive sequences makes A-EJ inherently less faithful than both primary pathways, but it provides a means for survival when other options are exhausted.
Genomic Instability Caused by A-EJ
The mechanism of A-EJ is inherently error-prone, making it a major contributor to genomic instability. The most common consequence is the loss of genetic material, known as a deletion. Deletions occur because the DNA sequence between the two aligned microhomologies is excised and lost from the genome during repair.
A-EJ can also lead to larger structural rearrangements, such as chromosomal translocations. A translocation happens when the broken end of one chromosome is mistakenly joined to the broken end of a different chromosome. The ability of A-EJ factors, particularly LIG3, to join disparate DNA ends increases the likelihood of these fusions.
These errors have profound biological consequences, contributing to the development and progression of various diseases. Dysregulation or overuse of A-EJ is observed in many human cancers, where the resulting chromosomal instability drives tumor evolution. Tumors with defects in HR or NHEJ become reliant on A-EJ for survival, making its components, like PARP-1 and LIG3, promising targets for new cancer therapies.