DNA Double-Strand Break Resection Occurs during Non-homologous End Joining in G1 but Is Distinct from Resection during Homologous Recombination.

Canonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis.

Nek1 Regulates Rad54 to Orchestrate Homologous Recombination and Replication Fork Stability.

Never-in-mitosis A-related kinase 1 (Nek1) has established roles in apoptosis and cell cycle regulation. We show that human Nek1 regulates homologous recombination (HR) by phosphorylating Rad54 at Ser572 in late G2 phase. Nek1 deficiency as well as expression of unphosphorylatable Rad54 (Rad54-S572A) cause unresolved Rad51 foci and confer a defect in HR. Phospho-mimic Rad54 (Rad54-S572E), in contrast, promotes HR and rescues the HR defect associated with Nek1 loss. Although expression of phospho-mimic Rad54 is beneficial for HR, it causes Rad51 removal from chromatin and degradation of stalled replication forks in S phase. Thus, G2-specific phosphorylation of Rad54 by Nek1 promotes Rad51 chromatin removal during HR in G2 phase, and its absence in S phase is required for replication fork stability. In summary, Nek1 regulates Rad51 removal to orchestrate HR and replication fork stability.

Catalytic and noncatalytic roles of the CtIP endonuclease in double-strand break end resection.

The carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) is known to function in 5' strand resection during homologous recombination, similar to the budding yeast Sae2 protein, but its role in this process is unclear. Here, we characterize recombinant human CtIP and find that it exhibits 5' flap endonuclease activity on branched DNA structures, independent of the MRN complex. Phosphorylation of CtIP at known damage-dependent sites and other sites is essential for its catalytic activity, although the S327 and T847 phosphorylation sites are dispensable. A catalytic mutant of CtIP that is deficient in endonuclease activity exhibits wild-type levels of homologous recombination at restriction enzyme-generated breaks but is deficient in processing topoisomerase adducts and radiation-induced breaks in human cells, suggesting that the nuclease activity of CtIP is specifically required for the removal of DNA adducts at sites of DNA breaks.

Factors determining DNA double-strand break repair pathway choice in G2 phase.

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.

ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2.

Homologous recombination (HR) and non-homologous end joining (NHEJ) represent distinct pathways for repairing DNA double-strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ-dependent process, which repairs a defined subset of radiation-induced DSBs in G1-phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB-repair pathway whereas HR is only essential for repair of approximately 15% of X- or gamma-ray-induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation-induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP-1, providing evidence that HR in G2 repairs heterochromatin-associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single-stranded DNA and Rad51 foci at radiation-induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ.

CtIP and MRN promote non-homologous end-joining of etoposide-induced DNA double-strand breaks in G1.

Topoisomerases class II (topoII) cleave and re-ligate the DNA double helix to allow the passage of an intact DNA strand through it. Chemotherapeutic drugs such as etoposide target topoII, interfere with the normal enzymatic cleavage/re-ligation reaction and create a DNA double-strand break (DSB) with the enzyme covalently bound to the 5'-end of the DNA. Such DSBs are repaired by one of the two major DSB repair pathways, non-homologous end-joining (NHEJ) or homologous recombination. However, prior to repair, the covalently bound topoII needs to be removed from the DNA end, a process requiring the MRX complex and ctp1 in fission yeast. CtIP, the mammalian ortholog of ctp1, is known to promote homologous recombination by resecting DSB ends. Here, we show that human cells arrested in G0/G1 repair etoposide-induced DSBs by NHEJ and, surprisingly, require the MRN complex (the ortholog of MRX) and CtIP. CtIP's function for repairing etoposide-induced DSBs by NHEJ in G0/G1 requires the Thr-847 but not the Ser-327 phosphorylation site, both of which are needed for resection during HR. This finding establishes that CtIP promotes NHEJ of etoposide-induced DSBs during G0/G1 phase with an end-processing function that is distinct to its resection function.

The G2/M checkpoint phosphatase cdc25C is located within centrosomes.

cdc25C is a phosphatase which regulates the activity of the mitosis promoting factor cyclin B/cdk1 by dephosphorylation, thus triggering G(2)/M transition. The activity and the sub-cellular localisation of cdc25C are regulated by phosphorylation. It is well accepted that cdc25C has to enter the nucleus to activate the cyclin B/cdk1 complex at G(2)/M transition. Here, we will show that cdc25C is located in the cytoplasm at defined dense structures, which according to immunofluorescence analysis, electron microscopy as well as biochemical subfractionation, are proven to be the centrosomes. Since cyclin B and cdk1 are also located at the centrosomes, this subfraction of cdc25C might participate in the control of the onset of mitosis suggesting a further role for cdc25C at the centrosomes.

Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1.

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein-interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku(-/-) mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847.

Role of ATM and the damage response mediator proteins 53BP1 and MDC1 in the maintenance of G(2)/M checkpoint arrest.

ATM-dependent initiation of the radiation-induced G(2)/M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G(2) phase are repaired by DNA nonhomologous end joining (NHEJ), while approximately 15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G(2)/M checkpoint is maintained in irradiated G(2) cells, in light of our current understanding of G(2) phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1(-/-) and MDC1(-/-) cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage.

gammaH2AX foci analysis for monitoring DNA double-strand break repair: strengths, limitations and optimization.

DNA double-strand breaks (DSBs) represent an important radiation-induced lesion and impaired DSB repair provides the best available correlation with radiosensitivity. Physical techniques for monitoring DSB repair require high, non-physiological doses and cannot reliably detect subtle defects. One outcome from extensive research into the DNA damage response is the observation that H2AX, a variant form of the histone H2A, undergoes extensive phosphorylation at the DSB, creating gammaH2AX foci that can be visualized by immunofluorescence. There is a close correlation between gammaH2AX foci and DSB numbers and between the rate of foci loss and DSB repair, providing a sensitive assay to monitor DSB repair in individual cells using physiological doses. However, gammaH2AX formation can occur at single-stranded DNA regions which arise during replication or repair and thus does not solely correlate with DSB formation. Here, we present and discuss evidence that following exposure to ionizing radiation, gammaH2AX foci analysis can provide a sensitive monitor of DSB formation and repair and describe techniques to optimize the analysis. We discuss the limitations and benefits of the technique, enabling the procedure to be optimally exploited but not misused.