Labeling of DNA replication sites and DNA fiber assays

Twenty-four h after transfection with GFP or Cdc45-GFP encoding vectors, HeLa CCL2 cells were grown in DMEM supplemented with 25 µM iodo-deoxyuridine (IdU, Sigma-Aldrich) for 30 or 45 min. After the first pulse labeling, the medium was removed and fresh DMEM containing 250 µM chloro-deoxyuridine (CldU, Sigma-Aldrich) was added for the second pulse label. Preparation of DNA spreads was done as described²¹ with minor modifications. Briefly, after labeling with halogenated nucleotide analogs and resuspension in PBS the cells were washed with ice-cold PBS and diluted to 5×10⁵ cells/ml. Approximately 1000 cells (2 µl) were lysed on glass slides by addition of 7 µl lysis buffer (200 mM Tris-HCl pH 7.4, 50 mM EDTA, 0.5 % SDS). DNA spreads were generated by slightly tilting the microscope slide and fixed using methanol/acetic acid (3:1) after air drying.

DNA spreads were denatured by incubation with 2.5 M HCl for 75 min at RT. The microscope slides were washed with PBS and blocked with TPB for 1 h. After incubation with a monoclonal rat anti-BrdU antibody (Clone BU1/75, 1:1000) in TPB for 1 h RT, which binds CldU but not IdU, the slides were washed twice with PBS and once with TPB and subsequently fixed using 4 % paraformaldehyde in PBS (10 min, RT), washed again twice with PBS and 3 times with TPB and incubated with goat anti-rat antibody conjugated with Alexa Fluor® 555 (Life Technologies, 1:500 in TPB) for 2 h at RT. DNA spreads were washed again as described above and incubated overnight at 4° C with a monoclonal mouse anti-BrdU antibody (Clone 44, 1:1500) which binds IdU, but not CldU. After washing, an Alexa Fluor® 488 conjugated goat anti-mouse antibody (Life Technologies, 1:500 in TPB) was added for 2 h at RT. Then the slides were extensively washed again and mounted. Pictures of fluorescent immunolabeled DNA spreads were taken using an Axio Imager.Z1 at 400-fold magnification.