Laser-Induced DSB Recruitment Dynamics

Generation of laser-induced DSB and recruitment of fluorescence protein-tagged proteins were performed with a SRS NL100 nitrogen MicroPoint system equipped to a Nikon Eclipse TE2000 spinning disk confocal microscope with Volocity software 6.3 (Perkin-Elmer)^{34 (link),64 (link)}. For treatment of CDK inhibitors, U2OS cells expressing GFP/YFP-tagged RECQL4 and YFP-MRE11 were pre-incubated with DMSO, 20 µM Roscovitine (Santa Cruz), 10 µM CDK1 inhibitor RO3306, 10 µM CDK2 inhibitor 2-III (EMD Millipore) for 4 h under standard cell culture conditions, and then subjected to micro point laser-irradiation. To study the function of CDK-dependent phosphorylation of Ser89 and Ser251 on RECQL4 recruitment to DSBs, GFP-tagged WT RECQL4, RQ4-2A, and RQ4-2D were expressed by transfecting the plasmids pEGFP-C1-RQ4Wt, pEGFP-C1-RQ4-2A, and pEGFP-C1-RQ4-2D into U2OS cells, respectively. For CDK 1and CDK2 inhibition, the U2OS cells were incubated with RO3306 and CDK2 inhibitor-III for 4 h before laser irradiation. The recruitment of these proteins was conducted as described above. The fluorescence intensity of the damaged area was measured with Volocity imaging software and normalized to that of a control area. The results are presented as mean ± s.e.m., and P -values were measured with Student’s t-test.