DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage, often leading to large chromosomal deletions. In mammalian cells, DSBs are primarily repaired by the non-homologous DNA end joining (NHEJ) pathway. This pathway involves proteins that recognize, resect, polymerize, and ligate DNA ends, allowing it to function on a wide range of DNA configurations, though often resulting in mutations. Recent studies have explored the involvement of different NHEJ proteins in repairing various DNA-end configurations. The review also discusses alternative pathways to DSB repair, such as alternative end joining (a-EJ) and single-strand annealing (SSA), and their relevance to human diseases. The authors highlight the importance of the cell cycle in determining which repair pathway is chosen, with NHEJ being dominant throughout the cell cycle, while a-EJ and SSA are favored during S and G2 phases. They also emphasize the role of specific proteins like Artemis, Pol μ, Pol λ, XRCC4, and DNA ligase IV in NHEJ and their interactions with other proteins. The review concludes by discussing future perspectives on the mechanisms and pathways involved in DSB repair, including the potential roles of a-EJ in meiosis and chromothripsis events.DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage, often leading to large chromosomal deletions. In mammalian cells, DSBs are primarily repaired by the non-homologous DNA end joining (NHEJ) pathway. This pathway involves proteins that recognize, resect, polymerize, and ligate DNA ends, allowing it to function on a wide range of DNA configurations, though often resulting in mutations. Recent studies have explored the involvement of different NHEJ proteins in repairing various DNA-end configurations. The review also discusses alternative pathways to DSB repair, such as alternative end joining (a-EJ) and single-strand annealing (SSA), and their relevance to human diseases. The authors highlight the importance of the cell cycle in determining which repair pathway is chosen, with NHEJ being dominant throughout the cell cycle, while a-EJ and SSA are favored during S and G2 phases. They also emphasize the role of specific proteins like Artemis, Pol μ, Pol λ, XRCC4, and DNA ligase IV in NHEJ and their interactions with other proteins. The review concludes by discussing future perspectives on the mechanisms and pathways involved in DSB repair, including the potential roles of a-EJ in meiosis and chromothripsis events.