The cell has developed two major pathways that are responsible for the repair of DNA DSB, homology directed repair (HDR) that is based homologous recombination and non-homologous end joining (NHEJ). The mechanism controlling the pathway choice for repair of DNA DSB in mammalian cells has not yet been clearly defined. However, it is thought that NHEJ, rather than HDR, is the predominant pathway for repair of DSB, particularly those induced by IR and other exogenous agents. A contributing factor to this hypothesis is that HDR requires a sister chromatid in close proximity that is used as a template in repair of the DSB and thus is restricted to S/G2. This mechanism has provided the nickname “errorfree” repair for HDR, as little to no loss of genetic material occurs, particularly if the template used is completely homologous. Importantly, specific DNA damage may be retained in HDR and require further repair or processing following initial HDR. Nondividing or cells not in S phase do not have a homologous donor, and as the majority of DNA damage from exogenous sources affects cells without a donor, NHEJ is thought to be responsible for the repair of most DSB caused by IR and other exogenous agents. Due to its ability to repair a DSB without a homologous template, NHEJ has been referred to as the “error-prone” pathway, as it is able to bring together two DNA ends that potentially have little to no homology at the site of the break. While in theory a simple mechanism, continuing research is showing that joining of two non-homologous DNA ends by NHEJ is in fact a sophisticated and complex mechanism of DNA repair.
NHEJ, found to be active throughout all phases of the cell cycle, is responsible for the joining of a DNA DSB. The pathway is most efficient in vitro at processing blunt termini that require no modification at the terminus prior to ligation. However, NHEJ is also proficient at joining two DNA ends that have nonhomologous overhang regions, and frequently this involves the removal or addition of nucleotides at the site of the break. Despite the term “non-homologous” end joining, it has been shown that there can be a greater tendency to join two broken ends that contain sequences with 1-4 nucleotides that are complementary, dubbed more recently as areas of microhomology. It is suggested that to align these ends of DNA at regions of microhomology, processing that results in the loss or addition of nucleotides must occur.
There are four specific steps in NHEJ; DNA termini recognition, bridging of the DNA ends also known as formation of the synaptic complex, DNA end processing, and finally DNA ligation. After a DSB occurs, the heterodimeric protein Ku, made up of 70 and 80 kDa subunits, binds to the end of the break. Once Ku is bound, it recruits the 465 kDa DNA-PK catalytic subunit (DNAPKcs). Together, these proteins make up a heterotrimeric complex called the DNAdependent protein kinase, or DNA-PK. The formation of this complex may aid in stabilizing the two DNA ends at the site of the break, forming a synaptic complex that secures the two ends together. The catalytic activity of DNA-PK is activated once bound to DNA, and this unique serine/threonine protein kinase phosphorylates downstream target proteins needed for completion of the pathway.
As mentioned earlier, IR does not frequently produce clean blunt-end breaks, and in fact regularly produces a number of complex breaks that contain DNA discontinuities at the terminus that require processing before proper ligation can occur. Artemis is the main nuclease known to process DNA termini in NHEJ, by degrading DNA single-strand overhangs with its 5’ exonuclease and 5’ or 3’ endonuclease activity. Cells containing defective Artemis are hypersensitive to radiation treatment. Polymerases responsible for adding bases at the termini include pol β, µ, and λ. Pol µ is of particular interest, as its concentration is increased in cells after IR exposure it is found in a complex with Ku and the Ligase IV/XRCC4 complex.
After processing of the DNA termini, DNA ligase IV is responsible for ligating the DSB. Ligase IV is able to ligate double-stranded DNA that has either compatible overhangs or blunt-ends, making it the perfect ligase for a repair pathway that does not require homology. DNA ligase IV is found in a complex with XRCC4, and the flexibility of this complex is apparent by the fact that the complex can ligate one strand even if the second strand can’t be ligated (perhaps because of a 5’ OH). XLF, a recently identified protein found to be involved in NHEJ just recently, was found to interact with Ligase IV/XRCC4 and found to be required for NHEJ and can complement DNA repair defects. Even more recent evidence has shown that XLF is in a complex with Ligase IV/XRCC4, and it is believed to be needed for stimulating the ligase activity of the complex.