The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination
Ultraviolet (UV) radiation-induced DNA lesions can be efficiently repaired by nucleotide excision repair (NER). However, NER is less effective during replication of UV-damaged chromosomes. In contrast, translesion DNA synthesis (TLS) and homologous recombination (HR) are capable of dealing with lesions in replicating DNA. The core HR protein in mammalian cells is the strand exchange protein RAD51, which is aided by numerous proteins, including RAD54. We used RAD54 as a cellular marker for HR to study the response of mammalian embryonic stem (ES) cells to UV irradiation. In contrast to yeast, ES cells lacking RAD54 are not UV sensitive. Here we show that the requirement for mammalian RAD54 is masked by active NER. By genetically inactivating NER and HR through disruption of the Xpa and Rad54 genes, respectively, we demonstrate the contribution of HR to chromosomal integrity upon UV irradiation. We demonstrate using chromosome fiber analysis at the individual replication fork level, that HR activity is important for the restart of DNA replication after induction of DNA damage by UV-light in NER-deficient cells. Furthermore, our data reveal RAD54-dependent and -independent contributions of HR to the cellular sensitivity to UV-light, and they uncover that RAD54 can compensate for the loss of TLS polymerase η with regard to UV-light sensitivity. In conclusion, we show that HR is important for the progression of UV-stalled replication forks in ES cells, and that protection of the fork is an interplay between HR and TLS.
|Keywords||Homologous recombination, Nucleotide excision repair, Replication fork stalling, Translesion synthesis, UV-light|
|Persistent URL||dx.doi.org/10.1016/j.dnarep.2011.08.006, hdl.handle.net/1765/34429|
|Journal||D N A Repair|
|Grant||This work was funded by the European Commission 7th Framework Programme; grant id fp7/259893 - The DNA damage response and breast cancer (DDRESPONSE)|
Eppink, B, Tafel, A, Hanada, K, van Drunen, E, Hickson, I.D, Essers, J, & Kanaar, R. (2011). The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination. D N A Repair, 10(11), 1095–1105. doi:10.1016/j.dnarep.2011.08.006