A novel DNA damage quantification platform enables high throughput screening for genes that impact DNA double strand breaks

Abstract

DNA is the blueprint of life, and the high fidelity transmission of genetic information from parent cells to progeny is essential for an organism's viability. However, our genomes are constantly being damaged by reactive molecules generated from cellular metabolic processes or introduced from the environment. The resulting DNA damage can alter the information encoded in DNA, and can interfere with the accurate transmission of genetic information when cells divide. The accumulation of cells with highly damaged or altered DNA within an organism can cause diseases, such as growth defects, aging and cancer. Fortunately, cells possess the capability to repair damaged DNA. Since DNA repair mechanisms can reverse the deleterious effects of DNA damage, they are important in disease prevention, and in particular play an important role in preventing cancer. DNA repair factors are also important targets for cancer therapies.

Tumor cells frequently harbor defects in DNA repair, rendering them vulnerable to DNA damage. Many cancer therapies exploit this vulnerability by treatment with DNA damaging agents. However, tumor cells can have differential DNA repair capacities based on the expression levels of various DNA repair genes. Thus, some cancer cells are variable in their response to chemotherapy and radiation. It is well established that inhibiting DNA repair can increase the efficacy of treatment. Therefore, it is critical to develop a better understanding of the network of genes that regulate DNA repair mechanisms both to understand susceptibility to cancer, and also in order to improve the outcomes of cancer therapy. DNA repair is a complex process that requires the coordination of many proteins to respond to specific classes of DNA damage. Many of the major proteins that directly participate in DNA repair pathways are well characterized.

However, recent research has indicated that the core DNA repair factors make up only a small fraction of the proteins that respond to DNA damage, suggesting that a large number of novel DNA repair factors have yet to be discovered and characterized. In this work, we leveraged the CometChip, a high-throughput DNA damage quantification assay, to screen thousands of genes for their ability to modulate DNA repair, by knocking them down with shRNAs. We first designed hardware for the CometChip to make it compatible with high-throughput robotics so as to reduce the amount of manual labor needed to execute our screen. We then exploited the ability of our assay to measure DNA damage at an unparalleled rate to screen an shRNA library targeting 2564 oncology-associated genes. We performed gene network analysis on the top candidate genes and found LATS2 to be a novel DNA repair factor. Further investigation revealed that LATS2 is a modulator of the homologous recombination repair pathway.

In addition, we merged our screen data with that from an assay that queries proteins for their ability to bind to DNA double strand breaks. Our results showed that we were able to identify known DNA repair factors via the intersection of the two datasets, and we pinpointed at least one other novel DNA repair gene for further investigation. Taken together, this work represents an advancement in the ability to discover novel DNA repair factors by large-scale parallel measurement of physical DNA damage in cells. Our technology enables high-throughput screening for DNA damage and repair factors faster than ever before, allowing for extensive studies of DNA damage and opening doors to the discovery of new genes and molecules that affect DNA repair.