Clusters of genomic damage induced by therapeutic and environmental ionizing radiation (IR)/radiomimetic drugs include double-strand breaks (DSBs) with nonligatable ends, and more abundant clusters of base/sugar oxidation products, abasic (AP) sites and single-strand breaks (SSBs). Highly cytotoxic DSBs, also formed during SSB replication, activate cell-cycle checkpoints and promote DSB repair (DSBR), which in mammalian genomes occurs via nonhomologous end joining (NHEJ) in all cells, and by error-free homologous recombination (HR) in S/G2 cells. Alternative end joining (Alt-EJ) also repairs DSBs, including those generated during the repair of bi-stranded non-DSB lesion clusters by DNA glycosylases (DGs), AP-endonuclease (APE1), and others via the BER/SSBR pathway. NHEJ-initiating Ku inhibits Alt-EJ which using microhomology- based SSBR is more error prone than NHEJ. Radiosensitivity caused by DG/APE1 deficiency and of HR negative tumors by inhibition of PARP-1-initiated SSBR indicates Alt-EJ/SSBR's significant contribution to radioresistance, which must be coordinated with NHEJ, the predominant DSBR pathway in mammals.BER prior to NHEJ would cause secondary DSBs which near preexisting DSBs would lead to larger deletions. This project's central hypothesis is that NHEJ precedes Alt-EJ/BER, coordinated by Ku (Ku70/80), which recruits DNA-PKcs at the DSB, followed by the DSB's end processing and re-ligation by DNA ligase4/XRCC4/XLF. Based on our preliminary studies showing that: (a) Ku present in DG/APE1 immunocomplexes (ICs) inhibits them;(b) the Ku IC from irradiated cells performs NHEJ of a novel linearized plasmid substrate with dirty ends, whose in-cell repair involves both NHEJ and Alt-EJ, we hypothesize that hnRNP-U, present in Ku IC only after irradiation, and phosphorylated by DNA-PK during NHEJ, relieves Ku inhibition, thus acting as a molecular switch for transition to Alt-EJ/BER which utilizes SSBR proteins and a distinct set of end-processing enzymes. We will test various facets of this comprehensive hypothesis by pursuing three aims:
Aim 1. To assess the contribution of Alt-EJ to radioresistance and its requirements in repairing radiation damage using reporter plasmid assays in-cell and in vitro, in parallel with analysis of cell genome repair, and to show that Alt-EJ/BER is additive to NHEJ in radioprotection.
Aim 2. To test the hypothesis that hnRNP-U and Ku together coordinate NHEJ and Alt-EJ via DNA-PK-mediated phosphorylation.
Aim 3. To test the hypothesis that NHEJ and Alt-EJ/BER proteins form repair-competent, dynamic complexes modulated by radiation/enediyne drugs involving physical interaction with Ku. We will characterize the end- processing enzymes and gap-filling DNA polymerase(s) for Alt-EJ. These studies will profoundly enhance our understanding of the repair of complex radiation-induced genomic damage, and of the contribution of Alt-EJ to radioresistance of tumors, and help identify novel therapeutic targets such as Ku for simultaneous radiosensitization of tumors and radioprotection of normal cells.

Public Health Relevance

IR exposure during therapy, diagnostic procedures or from the environment due to radiation accident or space travel causes mutagenic, cytotoxic and carcinogenic genomic damage. The long-term goal of this project is to illuminate the repair of radiation-induced genome damage, a process essential to restore genomic integrity. Achieving this goal could help improve therapeutic protocols for enhanced radiosensitization of tumor cells while better protecting healthy tissues from radiation damage.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-OTC-K (04))
Program Officer
Pelroy, Richard
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Methodist Hospital Research Institute
United States
Zip Code
Sengupta, Shiladitya; Yang, Chunying; Hegde, Muralidhar L et al. (2018) Acetylation of oxidized base repair-initiating NEIL1 DNA glycosylase required for chromatin-bound repair complex formation in the human genome increases cellular resistance to oxidative stress. DNA Repair (Amst) 66-67:1-10
Wang, Haibo; Dharmalingam, Prakash; Vasquez, Velmarini et al. (2017) Chronic oxidative damage together with genome repair deficiency in the neurons is a double whammy for neurodegeneration: Is damage response signaling a potential therapeutic target? Mech Ageing Dev 161:163-176
Wang, Haibo; Peng, Bin; Pandita, Raj K et al. (2017) Aurora kinase B dependent phosphorylation of 53BP1 is required for resolving merotelic kinetochore-microtubule attachment errors during mitosis. Oncotarget 8:48671-48687
Dutta, Arijit; Eckelmann, Bradley; Adhikari, Sanjay et al. (2017) Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex. Nucleic Acids Res 45:2585-2599
Hegde, Muralidhar L; Bohr, Vilhelm A; Mitra, Sankar (2017) DNA damage responses in central nervous system and age-associated neurodegeneration. Mech Ageing Dev 161:1-3
Yang, Chunying; Sengupta, Shiladitya; Hegde, Pavana M et al. (2017) Regulation of oxidized base damage repair by chromatin assembly factor 1 subunit A. Nucleic Acids Res 45:739-748
Hegde, Muralidhar L; Dutta, Arijit; Yang, Chunying et al. (2016) Scaffold attachment factor A (SAF-A) and Ku temporally regulate repair of radiation-induced clustered genome lesions. Oncotarget 7:54430-54444
Kont, Yasemin Saygideger; Dutta, Arijit; Mallisetty, Apurva et al. (2016) Depletion of tyrosyl DNA phosphodiesterase 2 activity enhances etoposide-mediated double-strand break formation and cell killing. DNA Repair (Amst) 43:38-47
Dutta, Arijit; Yang, Chunying; Sengupta, Shiladitya et al. (2015) New paradigms in the repair of oxidative damage in human genome: mechanisms ensuring repair of mutagenic base lesions during replication and involvement of accessory proteins. Cell Mol Life Sci 72:1679-98
Hegde, Pavana M; Dutta, Arijit; Sengupta, Shiladitya et al. (2015) The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome: DOMINANT NEGATIVE FUNCTION OF THE CTD. J Biol Chem 290:20919-33

Showing the most recent 10 out of 17 publications