Chapter 1: Introduction
1.9. DNA double-strand break signaling and repair
DSBs can be generated in various routes. In addition to “programmed” DSB formation during V(D)J recombination, class switch recombination and meiosis, DSB can also be formed by the “accidental” events, such as ionizing radiation, treatment of radiomimetic drugs like topoisomerase II poisons, or topoisomerase I poisons/crosslinking agents-mediated replication fork collapse (one-ended DSB).
DSB-induced DNA damage response43-45 DDR—DNA damage sensors, signal transducers and effectors.
When DSBs occur, the DSB sensor, MRE11/RAD50/NBS1 (MRN) complex, will bind to and recruit partially autoactivated ATM, one of phosphoinositide 3-kinase (PI3K)-like protein kinases, to the DSB site, then inducing fully activation of ATM46. Activated ATM phosphorylates numerous local substrates including histone variant H2A.X (γ-H2A.X) around DSB. γ-H2A.X can extend megabase pair distances from the DSB and trigger histone modifications around the DSB to increase DNA accessibility for downstream protein assembly.
DNA damage sensors transmit signals to transducers, which then amplify and
transduce signals to downstream effectors to result in cell cycle arrest. Chk2 and Chk1 are important transducers of ATM and ATR in response to DSB and DNA single-strand break (SSB) respectively. ATM/Chk2 and ATR/Chk1 pathways were historically thought to act in parallel with overlapping functions. However, more recently studies found these two pathways can be an upstream-downstream relationship, which explains why both ATM/Chk2 and ATR/Chk1 are activated and responsible for DSB-induced cell cycle arrest47-50. The proposed model45 (Appendix 3) suggests that upon DSBs, MRN complex recognizes the DSB and leads to recruitment and full activation of ATM. The activated ATM then can phosphorylate several effector kinases including Chk2, which results in G1 arrest through ATM-Chk2-p53-p21 pathway. Activated ATM can also promote the enrollment of CtIP to the site of DSB, where CtIP interacts with and stimulates the nuclease activity of MRE11 of MRN complex to start the end resection of DSB and generate short tracts of ssDNA. Other nucleases and helicases, such as Exo1 and BLM, further resect the ssDNA to form the more extensive regions for RPA to bind and initiate the homologous recombination-mediated DSB repair. Importantly, it has been noted that the exposed ssDNA regions act like SSBs to activate ATR/Chk1 pathway. Activated Chk1 can induce p53-independent S phase arrest via phosphorylation of Cdc25A for degradation51, or it can turn on G2/M checkpoint by phosphorylating and promoting Cdc25C for association with 14-3-3 proteins, preventing Cdc25C from activating mitotic Cdk1/cyclin B complex52, 53. Cell cycle arrest modulated by these transducer and effector kinases is important for the additional replication checkpoint responses (fork stabilization, inhibition of origin firing and S/M checkpoint) and the following DSB repair.
DNA double-strand break repair (Appendix 4)
Except for one-ended DSB which can only be repaired by homologous recombination (HR), two-ended DSB, such as IR or topoisomerase II-mediated DNA damage, can be repaired by either HR or non-homologous DNA end joining (NHEJ)54.
Homologous recombination (HR)
HR is an error-free DSB repair pathway. It is restricted to the late S and G2 phases of the cell cycle, where the homologous sequence located on the sister chromatid is available to serve as a donor template for repair of the damaged strand. The repair process of HR can be divided into three phases55, 56.
PhaseⅠ: Presynapsis
The ends of DSB are initially resected by MRN complex (Mre11-Rad50-Nbs1) and endonuclease CtIP complexed with BRCA1 in a 5’ to 3’ direction to generate short 3’-overhangs of single-strand DNAs (ssDNA). Further end resection is subsequently extended by Exo1, Dna2 and BLM to ensure the maintained resection. The resected ssDNA-ends are then coated by replication protein A (RPA) filaments, which keep ssDNAs unwound. Later, Rad51 together with other mediator proteins, such as BRCA2, Rad52 and Rad51 paralogs, replace RPA to form helical nucleoprotein filament on DNA.
Phase Ⅱ: Synapsis
Rad51 nucleofilaments promote the searching for homologous DNA sequences (sister chromatid in mitosis) similar to that of the 3’-overhangs, and catalyze strand invasion with the formation of displacement loop (D-loop).
Phase Ⅲ: Postsynapsis
After the successful strand invasion, DNA synthesis of the invading strand is carried out
by DNA polymerase using the donor sequence serving as a template. Depending on the different types of HR, D-loop can be resolved by dissociation of one of the invading strands (synthesis-dependent strand annealing pathway, SDSA), or through migrating double Holliday junction intermediate that is dissolved by BLM–RMI–TOP3 or cleaved by resolvases.
Non-homologous end joining (NHEJ)
NHEJ is an error-prone DNA double-strand break (DSB) repair pathway that is active throughout all cell cycle phases. Compared to HR pathway, NHEJ is a much faster repair process because it simply joins the DSB ends without ensuring the restoration of the original DNA sequence around the DSB site. It has been known that NHEJ is the predominant pathway to repair IR or topoisomerase Ⅱ inhibitors-induced DSBs in mammalian cells.
The repair process of NHEJ 55, 56:
The initiation of NHEJ begins from the binding of the Ku70/Ku80 heterodimer (Ku) to the exposed ends of DSB. Upon binding to DNA, Ku-DNA complex recruits and activates DNA-PKcs to the site of DSB. Activated DNA-PKcs has two important functions. It first thethers two opposing ends of DSB closly, and then recruits end-processing factors (for example Artemis, polynucleotide kinase/phosphatase (PNKP), AP endonuclease 1 (APE1) and tyrosyl–DNA phosphodiesterase 1 (TDP1)) to process the ends of DSB, which allows for religation by the XRCC4- XLF- LIG4 complex together with the polymerases λ and µ.