Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress.

ATR is an essential kinase activated in response to DNA-replication stress, with a known target being the RPA2 subunit of human replication protein A (RPA). We find that S33-RPA2 phosphorylation by ATR occurs primarily in the late-S and G2 phases, probably at sites of residual stalled DNA-replication forks, with S33-P-RPA2 contained within nuclear repair centers. Although cells in which endogenous RPA2 was ;replaced' with an RPA2 protein with mutations T21A and S33A (T21A/S33A-RPA) had normal levels of DNA replication under non-stress conditions, the mutant cells were severely deficient in the amount of DNA synthesis occurring during replication stress.

Mitotic crisis: the unmasking of a novel role for RPA.

Mitotic DNA damage is a constant threat to genomic integrity, yet understanding of the cellular responses to this stress remain incomplete. Recent work by Anantha et al. (2008; PNAS 105:12903-8) has found surprising evidence that RPA, the primary eukaryotic single-stranded DNA-binding protein, can stimulate the ability of cells to exit mitosis into a 2N G(1) phase.

RPA phosphorylation facilitates mitotic exit in response to mitotic DNA damage.

Human replication protein A (RPA) becomes phosphorylated on the RPA2 subunit by cyclin B-Cdc2 during mitosis, although the functional role of this modification is unclear. We find that this modification stimulates RPA2 to become hyperphosphorylated in response to mitotic DNA damage caused by bleomycin treatment. Cells in which endogenous RPA2 was replaced by a mutant subunit lacking both Cdc2 sites had a significant defect in mitotic release into a 2N G(1) phase after exposure to bleomycin.