Mammalian DNA double-strand break repair pathways
DNA DSBs are highly genotoxic lesions which, if left unrepaired, can result in genomic instability, cell death, and tumorigenesis. Mammalian cells consistently suffer from DSBs as result of exposure to genotoxic agents, irradiation, metabolic and oxidative stress, and as programmed events during the development of lymphocytes and germ cells. These lesions have to be precisely and efficiently repaired to maintain genomic integrity.
In mammalian cells, there are two major DNA DSB repair pathways: the Non-Homologous End-Joining (NHEJ) and the Homologous Recombination (HR) pathways. ATM has a role in both pathways and also contributes to the pathway choice by promoting end-resection, the first step of HR. Classical NHEJ (C-NHEJ) is a non-templated, error prone pathway that entails direct joining of DSBs. C-NHEJ functions throughout all cell cycle phases and is the predominant repair pathway in the G1 phase of the cell cycle when the homologous template is not usually available. Ku70/80 (Ku86 in human) protein complex binds to the ends of the DSB, initiate C-NHEJ, and recruit DNA-PKcs and Artemis endonuclease, which together processes the DNA ends (e.g. hairpin opening), among other functions. The formation of XLF/CERNUNNOS-XRCC4-LIG4 filaments on the DNA is thought to bridge the two participating DNA ends, where LIG4 catalyzes the ligation reaction to complete the joining process.
In contrast to C-NHEJ, HR is a template-mediated, error-free pathway of DSB repair. HR activity is largely restricted to S-phase and G2 phases of the cell cycle, when the sister-chromatid is available as a template. HR is initiated by 5’ to 3’ resection of the break site by the endo and exo-nucleases, including MRE11 with the help of CTIP (referred to as RBBP8 in the NCBI), and EXO1.
Resection of the ends results in the exposure of 3’ single-stranded DNA (ssDNA) overhangs that are coated by ssDNA binding protein RPA. RPA is hyper-phosphorylated by PIKKs, most notably at sites Ser4, Ser8, and Thr21, which primes their removal from the ssDNA ends by RAD52 and BRCA1/2, and replacement by RAD51 filaments. The coating of the ssDNA by Rad51 filaments primes the free DNA end for strand invasion- allowing the free DNA end to undergo homology search. In mitotic cells, the homologous template used for repair is primarily on the sister chromatid, while duing meiosis, homologous chromosomes are often the preferred template. The invasion and annealing of the donor DNA to its homologous sequence generates a Displacement loop (D-loop). The recapturing and reannealing of the invading strand to the original, non-template strand generate double Holliday Junctions (HJs), which are processed by BLM/Top3 complex or by structure-specific nucleases, such as MUS81 SLX1and GEN1.
Depending on the relative orientation of the cleavage at the two cross sites by the structure-specific endonuclease, HJ resolution can result in either a non-crossover event, where the chromosomal regions flanking the break sites are not exchanged, or a crossover where the resulting ends exchange sister chromatids. BLM helicase brings the two HJs in close proximity to allow Top3 to resolve it, which always results in non-crossover products. In cases when the second strand is not recaptured and the double HJs do not form, the invading strand can be displaced from the D-loop and re-anneal to the original, non-invading strand in a process known as synthesis-displacement strand annealing (SDSA) which does not result in a crossover. Alternatively, cells can also undergo Break-Induced Replication (BIR), where the non-invading second end is lost, and the invading strand can continue to synthesize the new strand until it reaches the end of the chromosome or is permanently displaced from the D-loop.
The recently emerged Microhomology-Mediated End Joining (MMEJ), also referred to as Alternative End-joining (AEJ), constitutes a third, less well-characterized DSB repair pathway. Although detailed mechanisms are still not well established, MMEJ relies on the presence of sequence microhomology flanking a DSB. These MH anneals to each when exposed upon resection, and effectively convert a DSB to two single strand breaks. As such, ligation during MMEJ is thought to be catalyzed by XRCC1/LIG3 and will result in the loss of the intervening sequences between the two MH sequences.