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Fig. 1. RPA34 is present as a hypophosphorylated form during the entire S phase. (A) Demembrated sperm nuclei (1000/µl) were incubated in a mitotic extract to which calcium was added to promote entry in S phase. DNA synthesis was followed by incorporation of [-³²P]dCTP. (B) Nuclei were isolated and treated with 0.3% Triton X-100, to collect the chromatin-bound (Chr) and nucleosolic-unbound (S) fractions, as described in Materials and Methods. The 309.112 polyclonal antibody (pAb) was used to reveal the RPA70 and RPA34, while dephosphorylated RPA34 was detected with the monoclonal antibody (mAb), as described in Materials and Methods and Fig. S1 (see supplementary material). (C) The binding of RPA to chromatin was analyzed during DNA replication and entry in mitosis induced by addition of 30 µg/ml of non-degradable B cyclin (cyclin B90). Nuclei were isolated and treated with 0.3% Triton X-100 to collect the chromatin-bound (Chr) and -unbound (S) nuclear fractions, as described in Materials and Methods. In mitotic or mitotic like-extracts (+ cyclin), the nuclear envelope does not form and the chromatin-associated (Chr) and cytoplasmic (Cyto) forms of RPA were analyzed. Fractions were analyzed by 12.5% SDS-PAGE and immunoblotted either with the 309.112 polyclonal antibody (pAb), or the monoclonal antibody (mAb) specific for dephosphorylated RPA34.

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