It is well known that ionizing radiation can break the double-stranded DNA in one or both the strands. Now a study by researchers at the Indian Institute of Science (IISc) Bengaluru have shown that regions of the genome rich in four-stranded DNA made of guanine nucleotide base — G-quadruplexes (G4-DNA) — are more resistant to irradiation. As a result, there are fewer DNA breaks seen in G-quadruplexes when exposed to radiation. The lower sensitivity to radiation was seen in studies carried out in vitro and inside cells.
The team led by Sathees Raghavan from the Department of Biochemistry at IISc found that contrary to the general notion that radiation-induced DNA breaks are random in nature and can occur throughout the genome, the breaks are sequence-dependent. Certain regions of the genome were found to be resistant to radiation with fewer strand breaks in the DNA, and these regions are rich in G-quadruplexes.
G-quadruplexes typically consist of three-guanine nucleotide base found together and repeated four times. When a guanine nucleotide gets repeated it tends to fold itself into a four-stranded DNA. There are 3.5 to 7 lakh G-quadruplexes in the human genome, and these are found in certain regions of the genome such as the telomeres that act as caps on either end of the chromosomes.
“When we exposed double-stranded DNA to radiation, the DNA was getting cut randomly. But those regions of the DNA containing G-quadruplexes had fewer breaks and so were protected from radiation,” says Prof. Raghavan. The results were published in the journal iScience.
Resilience of guanine
To test the resistance of guanine to radiation, the researchers started with single DNA strands. When single DNA strands made entirely of one of the four nucleotides — adenine, cytosine, guanine, or thymine — were exposed to gamma radiation, all except the strand made of guanine were sensitive to radiation.
In the case of a single DNA strand containing only thymine in one half and guanine in the other half, the guanine half alone showed better resistance to radiation.
But guanine loses its resistance when paired into double-strands and exposed to radiation. “We found guanine was resistant to radiation when present in a single strand but becoming sensitive to radiation when present in a double-strand form,” he says.
When the team made three double-stranded DNAs containing AT-rich, GC-rich and scrambled sequence and exposed to gamma radiation, all the three were equally susceptible to radiation. “When DNA is in normal double-helical form then guanine is equally susceptible to radiation unlike when it is in the G-quadruplex structure,” says Prof. Raghavan.
While dimethyl sulphate is able to cause DNA breaks when guanine is present in a double-strand, its ability to break DNA strands is compromised when guanine is present as G-quadruplex. “The position required for methylation [addition of methyl groups to the DNA] is occupied due to bonding, and so the G-quadruplex is resistant to dimethyl sulphate and no breaks are seen,” he says.
“Potassium chloride is essential for G-quadruplex formation. And in the presence of potassium chloride, the ability of dimethyl sulphate to induce cleavage in guanine is less, suggesting that guanine forms a G-quadruplex structure,” says Nitu Kumari from IISc and one of the first authors of the paper.
Inside the cells
The researchers tested the radiation resistance of G-quadruplex inside cells. The cells were exposed to 10 Gray gamma radiation and then stained with a fluorescent-labelled antibody to study if the telomere remains protected.
“There were fewer DNA breaks in the G-quadruplex present in telomeres compared with centromere [another part of the chromosome]. This suggests that G-quadruplex offers radioprotection inside the cell,” says Sumedha Dahal from IISc and another first author of the paper.
To reconfirm the protection offered by G-quadruplex, the researchers used an antibody that binds to the G-quadruplex structure and then irradiated the cell using 5, 10 and 20 Gray. “We examined the entire genome and found wherever G-quadruplex was present there was less DNA damage. Even at 20 gray gamma radiation no breaks were seen in the G-quadruplex structure unlike the rest of the genome,” says Susmita Kumari from IISc and another first author of the paper.
Public database
Data available in public database of whole genome sequencing of two cell lines post-irradiation were analysed for radioprotection offered by G-quadruplex by collaborators from Institute of Bioinformatics and Applied Biotechnology (IBAB). “There were fewer DNA breaks in the regions where G-quadruplex is present unlike the other regions of the genome,” says Bibha Choudhary.
To confirm the analysis of publicly available data, the researchers irradiated the genome and amplified the DNA sequences using a PCR. “All the genes that contained G-quadruplex structure showed less DNA breaks unlike other genes,” says Nitu Kumari.
Several other studies carried out too confirmed that G-quadruplex structure offered better protection to the DNA against radiation.
“Our study provides a new dimension to the role of altered DNA structures within the human genome, and helps study potential evolution of these structures. We also anticipate that our study will aid in exploring differential radiosensitivity across living organisms in correlation with the GC content of the genome,” says Prof. Raghavan.
