The question of how reproductive cells like sperm and eggs maintain their DNA integrity during development is at the heart of a new study by student researchers in the lab of molecular biologist Diana Libuda.
DNA damage occurs dozens or even hundreds of times within each individual developing sperm or egg cell, and the process occurs in organisms ranging from yeast cells to humans.
"The DNA repair pathway we studied is effectively perfect, in that it is able to resolve damage with no sign that there was ever damage there in the first place," said lead author Erik Toraason, a doctoral candidate in molecular biology. "Our research illuminates this seemingly invisible process and helps us better understand what particular proteins are doing in terms of preserving genome integrity."
He said the proteins they are studying in worms are also in humans, and mutations in those kinds of genes can have serious consequences.
The consequences of unrepaired breaks can include a group of genetic disorders called chromosomal breakage syndrome and other diseases. The research could someday inform treatment of cancer, infertility and complications due to aging.
The paper, "Meiotic DNA break repair can utilize homolog-independent chromatid templates in C. elegans," appeared in the journal Current Biology. Contributors include Libuda, the principal investigator, and UO researchers Anna Horacek, Cordell Clark, Marissa L. Glover, Victoria L. Adler and Alina Salagean - three of whom were undergraduate researchers at the time of the study - as well as Francesca Cole and Tolkappiyan Premkumar from the MD Anderson Cancer Center in Smithville, Texas.
The repair pathway that is the focus of the study occurs during meiosis, the process that ensures developing eggs and sperm contain the correct number of chromosomes. The pathway is difficult to study because it leaves no noticeable changes in the DNA as an indicator that it ever happened, Toraason said.
Using genetic tools, researchers developed an assay - an investigative procedure to measure activity - to generate and detect the repair of double-strand DNA breaks in C. elegans roundworms. Not only were investigators able to visualize the "invisible" repair events but they could also examine individual interactions of DNA molecules using sequencing analysis.
"This powerful assay enables us to detect when and how DNA breaks are repaired in developing eggs, and importantly, we can now actually start to figure out the mechanisms of these DNA repair events," said Libuda, a professor in the UO's Department of Biology and Institute of Molecular Biology. "We can put different genetic components into this assay and determine their function in maintaining our genetic information, or whether they have any function at all."
Among other things, researchers are hoping to gain a better understanding of how broken DNA molecules select from several nearly identical DNA templates to use for repair when a break occurs. The research has already revealed a specific gene that plays a novel role in the process of DNA repair.
The origins of the research project go back more than 10 years to Libuda's time as a postdoctoral fellow at Stanford University where she developed several earlier versions of the assay. The advent of new CRISPR-Cas9 genome editing technologies in the 2010s allowed Libuda to more precisely target her research. It led her to abandon earlier versions of the assay and discard years' worth of work.
CRISPR technology, which earned scientists Jennifer Doudna and Emmanuelle Charpentier the Nobel Prize in chemistry in 2020, has been described as "genetic scissors" that allow scientists to make precise edits to the genome.
"We can now make DNA breaks in exact locations in the genome and figure out how the breaks get repaired within a precise genomic context," Libuda said.
That's proven to be the case, and the assay is now assisting researchers around the world in determining the unknown functions of existing genetic components in DNA repair. Data from the assay could eventually be used in therapeutics to prevent birth defects and other problems that plague human health.
Libuda tapped a number of students for her project, all of whom have gone on to other organizations ranging from the Fred Hutchinson Cancer Research Center to the National Institutes of Health to the University of California, Berkeley. Libuda points to her own undergraduate research experiences at UCLA as being critical to her success as a scientist.
"One of the things that drew me to being at the UO is that it's a university that values undergraduate research and education as well as graduate education," Libuda said.
Salagean, a recent Clark Honors College graduate in biology, credits her experience in the Libuda Lab with preparing her to pursue a doctorate from the Massachusetts Institute of Technology starting in the fall.
"You come in to face this new research experience and you have no idea how to do all these techniques or what all of these different things mean," Salagean said. "Learning how to ask questions and to have confidence in asking those questions was just such a valuable experience."
The research was supported by the National Institutes of Health under award numbers R00HD076165, R35GM128890, T32GM007413, R25HD070817, R01HD098129, by the Knight Campus Undergraduate Scholars program and by an Advancing Science in America Foundation Award.
