Improving Understanding of Genetic Differences

Most humans are born with 46 chromosomes, 23 inherited from each parent. The chromosomes are housed in the nucleus of our cells and inside the chromosomes are genetic instructions known as DNA. But, as scientists discovered in 1964, some people also have another type of DNA called extrachromosomal circular DNA (eccDNA). It appears under a microscope like little circles floating around our chromosomes.

Investigators are just learning what role eccDNA plays in the body. Some evidence hints at eccDNA being involved in the development of cancer, for example. But to understand it, researchers need to get up close and personal with it. That's where the work by Hisashi Tanaka, MD, PhD, senior author of a paper published in the journal PNAS, and colleagues comes in.

Tanaka and fellow investigators Lila Mouakkad-Montoya, MS, and Michael Murata, PhD, at Cedars-Sinai invented a method to isolate circular DNA in human sperm and mouse tissues.

Tanaka and colleagues used a technique called centrifugation to separate larger DNA pieces from smaller DNA pieces in a test tube. After removing the larger pieces, they added an enzyme called exonuclease into the test tube to separate the eccDNA from other small DNA pieces. Exonuclease only breaks down linear DNA, so this is made it possible for the investigators to isolate the circular DNA from the linear DNA.

"Other researchers have used enzymes to expand eccDNA but when you do this, you lose the natural state and composition of the eccDNA," Tanaka said. "We were able to isolate the DNA from human tissue cells to study circular DNA in its natural state, which we call naïve small circular DNA (nscDNA).”

The investigators discovered that most eccDNA originates from regions where DNA of very similar sequences are next to each other and repeat themselves multiple times.

Scientists do not yet know for sure what causes eccDNA to appear in our cells. Previous research suggests it may arise when DNA is damaged. Some studies have found that in cancer cells, eccDNA can shuttle between being outside and inside of chromosomes, for example. Tanaka and colleagues propose an alternate mechanism: Tandemly repeated DNA sequences could create obstacles to DNA replication and trigger eccDNA.

"When cells divide, the nuclear membrane breaks down," Tanaka said. "eccDNA could leak out from the nucleus or get integrated somewhere in chromosomes."

This moving back and forth of eccDNA between chromosomes may contribute to genetic variation.  

"If eccDNA popped up from chromosome A and got integrated into chromosome B in sperm, a child born from that sperm would have a different genotype from the father,” Tanaka explained.

In upcoming studies, the investigators plan to compare eccDNA between cancer and healthy tissues and study what mechanisms might give rise to eccDNA.

Learn more about the Tanaka Lab.

Funding: Research reported in this study was supported by the National Cancer Institute under award numbers RO1 CA149385 and R03 CA188111-01A1, the U.S. Department of Defense under award number W81XWH-18-1-0058, Cedars-Sinai, the Margie and Robert E. Petersen Foundation, the Fashion Footwear Charitable Foundation of New York, the Avon Foundation and Associates for Breast and Prostate Cancer Studies.