Repair Of IR-induced DNA Double-strand Breaks Research Articles

RNA binding protein hnRNP‐U coordinates repair of radiation‐induced damage clusters in human genome via its Ser59 phosphorylation (736.8)

Ionizing radiation (IR), widely used in diagnostic procedures and cancer therapy, induces complex clusters of damage in the genome that include DNA double‐strand breaks (DSB) and much higher levels of closely spaced oxidized bases and single strand‐breaks (SSB). While overt DSBs are primarily repaired via non‐homologous end joining (NHEJ), oxidized bases and SSBs are repaired via base excision repair (BER)/ SSB repair pathway. However, how these multiple repair pathways are co‐ordinated at IR‐induced damage clusters is poorly understood. Here we demonstrate temporal control of NHEJ vs. BER, co‐ordinated by RNA binding protein hnRNP‐U, which was recently implicated in regulating BER for enhancing the activities of NEIL1/NEIL2 glycosylases. We found Ku, a key NHEJ protein inhibits early BER proteins (NEILs, OGG1 & APE1) at the damage clusters, preventing repair of oxidized bases at close proximity. hnRNP‐U, also recruited at the repair site cannot overcome Ku inhibition initially due to its phosphorylated state (at S59 by DNA‐PK in response to IR). Subsequently, hnRNP‐U dephosphorylated after NHEJ completion relieves Ku inhibition and facilitates BER initiation. This differential ability of unmodified vs. S59‐P hnRNP‐U is due to its phosphorylation‐dependent decrease in affinity for the DNA glycosylases. These studies provide a molecular mechanism for sequential repair of IR‐induced DSBs followed by BER of proximal oxidized bases, where hnRNP‐U acts as a ‘molecular switch’. We propose that such hierarchical repair of IR‐induced damage is important in maintaining genomic fidelity.Grant Funding Source: Supported by USPHS grant R01 CA158910 and HMRI

BMS-345541 Sensitizes MCF-7 Breast Cancer Cells to Ionizing Radiation by Selective Inhibition of Homologous Recombinational Repair of DNA Double-Strand Breaks

Our study was to elucidate the mechanisms whereby BMS-345541 (BMS, a specific IκB kinase β inhibitor) inhibits the repair of DNA double-strand breaks (DSBs) and evaluate whether BMS can sensitize MCF-7 breast cancer cells (MCF-7 cells) to ionizing radiation (IR) in an apoptosis-independent manner. In this study, MCF-7 cells were exposed to IR in vitro and in vivo with or without pretreatment of BMS. The effects of BMS on the repair of IR-induced DSBs by homologous recombination (HR) and non-homologous end-joining (NHEJ) were analyzed by the DR-GFP and EJ5-GFP reporter assays and IR-induced γ-H2AX, 53BP1, Brca1 and Rad51 foci assays. The mechanisms by which BMS inhibits HR were examined by microarray analysis and quantitative reverse transcription PCR. The effects of BMS on the sensitivity of MCF-7 cells to IR were determined by MTT and clonogenic assays in vitro and tumor growth inhibition in vivo in a xenograft mouse model. The results showed that BMS selectively inhibited HR repair of DSBs in MCF-7 cells, most likely by down-regulation of several genes that participate in HR. This resulted in a significant increase in the DNA damage response that sensitizes MCF-7 cells to IR-induced cell death in an apoptosis-independent manner. Furthermore, BMS treatment sensitized MCF-7 xenograft tumors to radiation therapy in vivo in an association with a significant delay in the repair of IR-induced DSBs. These data suggest that BMS is a novel HR inhibitor that has the potential to be used as a radiosensitizer to increase the responsiveness of cancer to radiotherapy.

IKKβ Regulates the Repair of DNA Double-Strand Breaks Induced by Ionizing Radiation in MCF-7 Breast Cancer Cells

Activation of the IKK-NFκB pathway increases the resistance of cancer cells to ionizing radiation (IR). This effect has been largely attributed to the induction of anti-apoptotic proteins by NFκB. Since efficient repair of DNA double strand breaks (DSBs) is required for the clonogenic survival of irradiated cells, we investigated if activation of the IKK-NFκB pathway also regulates DSB repair to promote cell survival after IR. We found that inhibition of the IKK-NFκB pathway with a specific IKKβ inhibitor significantly reduced the repair of IR-induced DSBs in MCF-7 cells. The repair of DSBs was also significantly inhibited by silencing IKKβ expression with IKKβ shRNA. However, down-regulation of IKKα expression with IKKα shRNA had no significant effect on the repair of IR-induced DSBs. Similar findings were also observed in IKKα and/or IKKβ knockout mouse embryonic fibroblasts (MEFs). More importantly, inhibition of IKKβ with an inhibitor or down-regulation of IKKβ with IKKβ shRNA sensitized MCF-7 cells to IR-induced clonogenic cell death. DSB repair function and resistance to IR were completely restored by IKKβ reconstitution in IKKβ-knockdown MCF-7 cells. These findings demonstrate that IKKβ can regulate the repair of DSBs, a previously undescribed and important IKKβ kinase function; and inhibition of DSB repair may contribute to cance cell radiosensitization induced by IKKβ inhibition. As such, specific inhibition of IKKβ may represents a more effective approach to sensitize cancer cells to radiotherapy.

Hematopoietic Stem Cells Repair Ionizing Radiation-Induced DNA Double Strand Breaks in a Cell Cycle-Dependent Manner.

Abstract 3241Poster Board III-178Mice with mutations in various DNA repair genes exhibit accelerated aging due to hematopoietic stem cell (HSC) premature exhaustion, indicating that DNA repair is crucial for the maintenance of HSC self-renewal and hematopoietic function. In addition, some of these mutated mice are highly susceptible to the development of leukemia and lymphoma due to an increase in genomic instability in HSCs. However, how HSCs respond to genotoxic stress and repair DNA damage have not been well established and thus, were investigated in the present study using a mouse model. Specifically, DNA damage and repair were analyzed by gH2AX immunofluorescent staining and neutral comet assay to quantify IR-induced DNA double strand breaks (DSBs) in HSCs (Lin- c-kit+ Sca1+ cells or LKS+ cells) and hematopoietic progenitor cells (HPCs; Lin- c-kit+ Sca1- cells or LKS- cells) isolated from adult mouse bone marrow (BM) after they were exposed to ionizing radiation (IR). The results showed that exposure to IR induced a similar number of DSBs in HSCs and hematopoietic progenitor cells (HPCs) isolated from adult mouse BM. However, HPCs repaired the damage within 6 h after IR, whereas more than 50% DSBs were unrepaired by HSCs even at 24h after IR, indicating that HSCs are highly deficient in repair of IR-induced DSBs. The deficient DSBs repair in HSCs is attributable to their quiescence, as sorted quiescent Pyronin Ylow HSCs were more deficient in repairing the damage than cycling Pyronin Yhigh HSCs. This suggestion is further supported by the observations that proliferating HSCs such as fetal liver HSCs and HSCs isolated from 5-FU-treated adult mouse BM repaired the damage as efficiently as HPCs. In addition, incubation of quiescent Pyronin Ylow HSCs from adult BM with stem cell factor and thrombopoietin for 48 h stimulated the cell cycle entry and DNA damage repair function. These findings indicate that stimulation of cell cycling can promote HSCs to repair DNA damage. The difference in repair of IR-induced DSBs between quiescent and cycling HSCs is not because they express different levels of key proteins (such as Ku70, Ku80, DNA-PKcs, Lig4, XRCC4, Dclre1c, Nhej1, Brac-1, Brac-2, MRE11a, Nbs1, Rad50, Rad51, and ATM) involved in non-homologous end joining (NHEJ) and homologous recombination (HR). Instead, quiescent HSCs exhibited an insignificant activation of DNA-PK and minimal formation of XRCC4 and Rad51 foci after exposure to IR, suggesting that quiescent HSCs are deficient in DSBs repair through the NHEJ and HR pathways. However, quiescent HSCs exhibited similar levels of phosphorylation of ATM and p53 after IR compared to cycling HSCs and HPCs, indicating that quiescent HSCs are proficient in sensing DNA damage to initiate DNA damage responses. These findings provide crucial insights into how HSCs respond to and repair DNA damage, which could significantly advance our understanding on how HSCs maintain their genomic stability. DisclosuresNo relevant conflicts of interest to declare.

Poly(ADP-Ribose) Polymerase-1 and DNA-Dependent Protein Kinase Have Equivalent Roles in Double Strand Break Repair Following Ionizing Radiation

Radiation-induced DNA double strand breaks (DSBs) are predominantly repaired by nonhomologous end joining (NHEJ), involving DNA-dependent protein kinase (DNA-PK). Poly(ADP-ribose) polymerase-1 (PARP-1), well characterized for its role in single strand break repair, may also facilitate DSB repair. We investigated the activation of these enzymes by differing DNA ends and their interaction in the cellular response to ionizing radiation (IR). The effect of PARP and DNA-PK inhibitors (KU-0058684 and NU7441) on repair of IR-induced DSBs was investigated in DNA-PK and PARP-1 proficient and deficient cells by measuring gammaH2AX foci and neutral comets. Complementary in vitro enzyme kinetics assays demonstrated the affinities of DNA-PK and PARP-1 for DSBs with varying DNA termini. DNA-PK and PARP-1 both promoted the fast phase of resolution of IR-induced DSBs in cells. Inactivation of both enzymes was not additive, suggesting that PARP-1 and DNA-PK cooperate within the same pathway to promote DSB repair. The affinities of the two enzymes for oligonucleotides with blunt, 3' GGG or 5' GGG overhanging termini were similar and overlapping (K(d)(app) = 2.6-6.4nM for DNA-PK; 1.7-4.5nM for PARP-1). DNA-PK showed a slightly greater affinity for overhanging DNA and was significantly more efficient when activated by a 5' GGG overhang. PARP-1 had a preference for blunt-ended DNA and required a separate factor for efficient stimulation by a 5' GGG overhang. DNA-PK and PARP-1 are both required in a pathway facilitating the fast phase of DNA DSB repair.

DNA-PK and ATM phosphorylation sites in XLF/Cernunnos are not required for repair of DNA double strand breaks

Nonhomologous end joining (NHEJ) is the major pathway for the repair of DNA double strand breaks (DSBs) in human cells. NHEJ requires the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku70, Ku80, XRCC4, DNA ligase IV and Artemis, as well as DNA polymerases μ and λ and polynucleotide kinase. Recent studies have identified an additional participant, XLF, for XRCC4-like factor (also called Cernunnos), which interacts with the XRCC4–DNA ligase IV complex and stimulates its activity in vitro, however, its precise role in the DNA damage response is not fully understood. Since the protein kinase activity of DNA-PKcs is required for NHEJ, we asked whether XLF might be a physiological target of DNA-PK. Here, we have identified two major in vitro DNA-PK phosphorylation sites in the C-terminal region of XLF, serines 245 and 251. We show that these represent the major phosphorylation sites in XLF in vivo and that serine 245 is phosphorylated in vivo by DNA-PK, while serine 251 is phosphorylated by Ataxia-Telangiectasia Mutated (ATM). However, phosphorylation of XLF did not have a significant effect on the ability of XLF to interact with DNA in vitro or its recruitment to laser-induced DSBs in vivo. Similarly, XLF in which the identified in vivo phosphorylation sites were mutated to alanine was able to complement the DSB repair defect as well as radiation sensitivity in XLF-deficient 2BN cells. We conclude that phosphorylation of XLF at these sites does not play a major role in the repair of IR-induced DSBs in vivo.

Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends.

Non-homologous end joining (NHEJ) is one of the primary pathways for the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) in mammalian cells. Proteins required for NHEJ include the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku, XRCC4 and DNA ligase IV. Current models predict that DNA-PKcs, Ku, XRCC4 and DNA ligase IV assemble at DSBs and that the protein kinase activity of DNA-PKcs is essential for NHEJ-mediated repair of DSBs in vivo. We previously identified a cluster of autophosphorylation sites between amino acids 2609 and 2647 of DNA-PKcs. Cells expressing DNA-PKcs in which these autophosphorylation sites have been mutated to alanine are highly radiosensitive and defective in their ability to repair DSBs in the context of extrachromosomal assays. Here, we show that cells expressing DNA-PKcs with mutated autophosphorylation sites are also defective in the repair of IR-induced DSBs in the context of chromatin. Purified DNA-PKcs proteins containing serine/threonine to alanine or aspartate mutations at this cluster of autophosphorylation sites were indistinguishable from wild-type (wt) protein with respect to protein kinase activity. However, mutant DNA-PKcs proteins were defective relative to wt DNA-PKcs with respect to their ability to support T4 DNA ligase-mediated intermolecular ligation of DNA ends. We propose that autophosphorylation of DNA-PKcs at this cluster of sites is important for remodeling of DNA-PK complexes at DNA ends prior to DNA end joining.