Replication protein A and the Mre11.Rad50.Nbs1 complex co-localize and interact at sites of stalled replication forks

Jacob G Robison, James Elliott, Kathleen Dixon, Gregory G Oakley
Journal of Biological Chemistry 2004 August 13, 279 (33): 34802-10
In response to replicative stress, cells relocate and activate DNA repair and cell cycle arrest proteins such as replication protein A (RPA, a three subunit protein complex required for DNA replication and DNA repair) and the MRN complex (consisting of Mre11, Rad50, and Nbs1; involved in DNA double-strand break repair). There is increasing evidence that both of these complexes play a central role in DNA damage recognition, activation of cell cycle checkpoints, and DNA repair pathways. Here we demonstrate that RPA and the MRN complex co-localize to discrete foci and interact in response to DNA replication fork blockage induced by hydroxyurea (HU) or ultraviolet light (UV). Members of both RPA and the MRN complexes become phosphorylated during S-phase and in response to replication fork blockage. Analysis of RPA and Mre11 in fractionated lysates (cytoplasmic/nucleoplasmic, chromatin-bound, and nuclear matrix fractions) showed increased hyperphosphorylated-RPA and phosphorylated-Mre11 in the chromatin-bound fractions. HU and UV treatment also led to co-localization of hyperphosphorylated RPA and Mre11 to discrete detergent-resistant nuclear foci. An interaction between RPA and Mre11 was demonstrated by co-immunoprecipitation of both protein complexes with anti-Mre11, anti-Rad50, anti-NBS1, or anti-RPA antibodies. Phosphatase treatment with calf intestinal phosphatase or lambda-phosphatase not only de-phosphorylated RPA and Mre11 but also abrogated the ability of RPA and the MRN complex to co-immunoprecipitate. Together, these data demonstrate that RPA and the MRN complex co-localize and interact after HU- or UV-induced replication stress and suggest that protein phosphorylation may play a role in this interaction.

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