G4 DNA present at human telomeric DNA contributes toward reduced sensitivity to γ-radiation induced oxidative damage, but not bulky adduct formation

Abstract

Purpose

DNA, the hereditary material of a human cell generally exists as Watson-Crick base paired double-stranded B-DNA. Studies suggest that DNA can also exist in non-B forms, such as four stranded G-quadruplexes (G4 DNA). Recently, our studies revealed that the regions of DNA that can fold into G-quadruplex structures are less sensitive to ionizing radiation (IR) compared to B-DNA. Importantly, we reported that the planar G-quartet of a G4 structure is shielded from radiation induced DNA breaks, while the single- and double-stranded DNA regions remained susceptible. Thus, in the present study, we investigate whether telomeric repeat DNA present at the end of telomere, known to fold into G4 DNA can protect from radiation induced damages including strand breaks, oxidation of purines and bulky adduct formation on DNA.

Materials and methods

For plasmid irradiation assay, plasmids containing human telomeric repeat DNA sequence TTAGGG (0.8 kb or 1.8 kb) were irradiated with increasing doses of IR along with appropriate control plasmids and products were resolved on 1% agarose gel. Radioprotection was evaluated based on extent of conversion of supercoiled to nicked or linear forms of the DNA following irradiation. Formation of G-quadruplex structure on supercoiled DNA was evaluated based on circular dichroism (CD) spectroscopy studies. Cleavage of radiation induced oxidative damage and extent of formation of nicks was further evaluated using base and nucleotide excision repair proteins.

Results

Results from CD studies showed that the plasmid DNA harboring human telomeric repeats (TTAGGG) can fold into G-quadruplex DNA structures. Further, results showed that human telomeric repeat sequence when present on a plasmid can protect the plasmid DNA against IR induced DNA strand breaks, unlike control plasmids bearing random DNA sequence.

Conclusions

Human telomeric repeat sequence when present on plasmids can fold into G-quadruplex DNA structures, and can protect the DNA against IR induced DNA strand breaks and oxidative damage. These results in conjunction with our previous studies suggest that telomeric repeat sequence imparts less sensitivity to IR and thus telomeres of chromosomes are protected from radiation.

Keywords:

• Non-B DNA
• G-quadruplex DNA
• radioresistance
• double-stranded break
• ionizing radiation
• radiosensitivity

Acknowledgements

The authors thank Urbi Roy, Dipayan Ghosh and other members of SCR laboratory for critical reading of the manuscript.

Author contributions

SCR, UV, UR, SS, IK, SK and SV designed the experiments; UR, SS, IK, SK, VG, SV and NK conducted the experiments. SCR, UV, UR, IK, SV and SK interpreted the results and wrote the manuscript.

Disclosure statement

Authors disclose that there is no conflict of interest.

Additional information

Funding

This work was supported by grants from DAE [21/01/2016-BRNS/35074], DBT [BT/PR13458/COE/34/33/2015], IISc-DBT [BT/PR27952-INF/22/212/2018] for SCR; DBT [BT/PR13522/CoE/34/27/2015] and The Jamsetji Tata Trust, India for UV. UR is supported by Research Associateship from IISc, Bangalore, SS by Junior Research fellowship (JRF) from DBT, IK by Research Associateship from DBT-COE and SK by JRF from IISc, Bangalore.

Notes on contributors

Ujjayinee Ray

Ujjayinee Ray, PhD, is a Research Associate in the Department of Biochemistry at Indian Institute of Science, Bangalore.

Shivangi Sharma

Shivangi Sharma, MSc, is a Junior Research Fellow from Department of Biotechnology, Ministry of Science and Technology, working in the Department of Biochemistry at Indian Institute of Science, Bangalore.

Indu Kapoor

Indu Kapoor, PhD, is a Research Associate from Department of Biotechnology, Ministry of Science and Technology, working in the Department of Microbiology and Cell Biology at Indian Institute of Science, Bangalore.

Susmita Kumari

Susmita Kumari, MSc, is a Junior Research Fellow in the Department of Biochemistry at Indian Institute of Science, Bangalore.

Vidya Gopalakrishnan

Vidya Gopalakrishnan, PhD, is an Assistant Professor at the Department of Zoology, St. Joseph's College, Irinjalakuda, Kerala, India.

Supriya V. Vartak

Supriya V. Vartak, PhD, is a Research Associate in Department of Biochemistry at Indian Institute of Science, Bangalore.

Nitu Kumari

Nitu Kumari, MSc, is a Senior Research Fellow in the Department of Biochemistry at Indian Institute of Science, Bangalore.

Umesh Varshney

Umesh Varshney, PhD, is a Professor at Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, working on the mechanistic aspects of protein synthesis and DNA repair using E. coli and Mycobacteria as model systems.

Sathees C. Raghavan

Sathees C. Raghavan, PhD, is a Professor at Department of Biochemistry, Indian Institute of Science, Bangalore, working on understanding the mechanism and regulation of DNA double-strand break repair, generation of chromosomal translocation and its role in oncogenesis, and evaluation of novel small molecule inhibitors in cancer treatment.