As progression through mitosis with even a single unrepaired DSB will lead to the loss of genetic information, the DDR system is extremely sensitive. Protein kinases ATM, ATR and DNA-PK-members of the phosphatidylinositol 3-kinase-related kinase (PIKK) superfamily ( 14)-play a central role in this process.

On the other hand, there are numerous differences in the biology between GT-proficient and GT-refractory cells, which may include the mechanism of delivery of the exogenous DNA, cellular response to its presence, its persistence and processing within the cell.ĭSBs in genomic DNA are critical lesions for dividing cells, and elaborate signaling systems, the DNA damage response (DDR), have evolved to detect them and delay the cell cycle progression until they are repaired ( 12, 13). However, HR proteins are ubiquitously expressed in animal tissues and are essential for the viability of proliferating cells. Yeast relies primarily on HR for DSB repair, whereas in higher eukarya, homology-independent mechanisms play a more prominent role. Part of the difference can be attributed to the relative activities of the DNA repair systems that are responsible for stable integration of the exogenous DNA. Why exactly GT is more efficient in some cells than the others is unclear. The technique is efficient and straightforward in model yeast species ( Saccharomyces cerevisiae and Schizosaccharomyces pombe) ( 5), some protists ( 6), a plant species ( 7) and also practical in several vertebrate cell lines ( 8–11), of which mouse embryonic stem (ES) cells are arguably the most important, as they allowed and popularized performing reverse genetics studies in a mammalian model organism. Gene targeting (GT) by homologous recombination (HR) is a genetic tool of unrivaled power and flexibility ( 1, 2) that was instrumental in the development of the famous double-strand break (DSB) model of HR ( 3, 4). Our results suggest that recombination pathways dependent on extensive homology search are caffeine-sensitive and stress the importance of considering direct checkpoint-independent mechanisms in the interpretation of the effects of caffeine on DNA repair. Biochemical and structural biology experiments revealed that caffeine interfered with a pivotal step in homologous recombination, homologous joint molecule formation, through increasing interactions of the RAD51 nucleoprotein filament with non-homologous DNA. We used chemical inhibitors and genetically deficient mouse embryonic stem cell lines to study the role of DNA damage response in stable integration of the transfected DNA and found that caffeine rapidly, efficiently and reversibly inhibited homologous integration of the transfected DNA as measured by several homologous recombination-mediated gene-targeting assays. Caffeine is a widely used inhibitor of the protein kinases that play a central role in the DNA damage response.

Caffeine molecule