Abstract
Caffeine is a widely used inhibitor of the protein kinases that play a central role in the DNA damage response. 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. 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. 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.
Highlights
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]
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
As several independent approaches did not lend support for the hypothesis that the strong inhibition of GT by caffeine, which we demonstrated in three different assays, is mediated by the DNA damage response (DDR), and other indirect pharmacological effects of caffeine could not be linked to GT inhibition either, we proceeded to test the possibility that caffeine affects the HR reaction directly
Summary
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]. The technique is efficient and straightforward in model yeast species (Saccharomyces cerevisiae and Schizosaccharomyces pombe) [5], some protists [6], a plant species [7] and practical in several vertebrate cell lines [8,9,10,11], 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. 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. Yeast relies primarily on HR for DSB repair, whereas in higher eukarya, homology-independent mechanisms play a more prominent role. 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
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