Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein, required for cellular DNA replication, repair, and recombination. In human cells, RPA is phosphorylated during the S and G 2 phases of the cell cycle and also in response to ionizing or ultraviolet radiation. Saccharomyces cerevisiae exhibits a similar pattern of cell cycle-regulated RPA phosphorylation, and our studies indicate that the radiation-induced reactions occur in yeast as well. We have examined yeast RPA phosphorylation during the normal cell cycle and in response to environmental insult, and have demonstrated that the checkpoint gene MEC1 is required for the reaction under all conditions tested. Through examination of several checkpoint mutants, we have placed RPA phosphorylation in a novel pathway of the DNA damage response. MEC1 is similar in sequence to human ATM, the gene mutated in patients with ataxia-telangiectasia (A-T). A-T cells are deficient in multiple checkpoint pathways and are hypersensitive to killing by ionizing radiation. Because A-T cells exhibit a delay in ionizing radiation-induced RPA phosphorylation, our results indicate a functional similarity between MEC1 and ATM, and suggest that RPA phosphorylation is involved in a conserved eukaryotic DNA damage-response pathway defective in A-T.

The 32-kDa subunit of replication protein A (RPA) is phosphorylated during the S phase of the cell cycle in vivo and during simian virus 40 DNA replication in vitro. To explore the functional significance of this modification, we purified a HeLa cell protein kinase that phosphorylates RPA in the presence of single-stranded DNA. By several criteria we identified the purified enzyme as a form of the DNA-activated protein kinase (DNA-PK), a previously described high molecular weight protein kinase that is capable of phosphorylating a number of nuclear DNA binding proteins. Phosphorylation of RPA by DNA-PK is stimulated by natural single-stranded DNAs but not by homopolymers lacking secondary structure. Studies with the simian virus 40 model system indicate that DNA-PK is required for DNA-replication-dependent RPA phosphorylation. Depletion of the kinase activity, however, has no effect on the extent of DNA replication in vitro. Our data support a model in which phosphorylation of RPA by DNA-PK is activated by formation of replication intermediates containing single-and double-stranded regions. This event may be involved in a signaling mechanism that coordinates DNA replication with the cell cycle.DNA replication in eukaryotic cells is a tightly regulated process. During the S phase of the cell cycle, DNA synthesis is initiated at multiple origins of replication distributed along the chromosomal DNA. Activation of each replication origin occurs once and only once, ensuring that exactly two copies of each segment of chromosomal DNA are produced during each cell cycle. The timing of initiation is also precisely controlled and is sensitive to both environmental and cellular factors. If DNA replication is blocked by inhibitors or the template is damaged by radiation or other factors, a signal is generated that delays cell cycle progression. This mechanism presumably functions to allow time for the cell to deal with the abnormal condition before attempting mitosis.Direct biochemical studies on the regulation of chromosomal replication have been difficult because of the complexity of the cellular genome. However, simian virus 40 (SV40) has provided a useful approach to this problem, since viral DNA replication is almost completely dependent upon cellular replication machinery (1-3). Analysis of viral DNA replication in vitro has led to the identification of a number of cellular replication proteins, and several lines of evidence suggest that initiation of viral DNA replication may be regulated by mechanisms similar in at least some respects to those that control cellular DNA replication during the cell cycle (4).One cellular replication protein identified by biochemical analysis of the SV40 system is replication protein A (RPA), a heterotrimeric single-stranded DNA (ssDNA)-binding protein that is required during both the initiation and elongation phases ofDNA replication (5-7). The ssDNA binding activity of RPA resides in the 70-kDa subunit (RPA70) and is required for the unwinding of the duplex at the SV40 origin...

The checkpoint mechanisms that delay cell cycle progression in response to DNA damage or inhibition of DNA replication are necessary for maintenance of genetic stability in eukaryotic cells. Potential targets of checkpoint-mediated regulation include proteins directly involved in DNA metabolism, such as the cellular single-stranded DNA (ssDNA) binding protein, replication protein A (RPA). Studies in Saccharomyces cerevisiae have revealed that the RPA large subunit (Rfa1p) is involved in the G1 and S phase DNA damage checkpoints. We now demonstrate that Rfa1p is phosphorylated in response to various forms of genotoxic stress, including radiation and hydroxyurea exposure, and further show that phosphorylation of Rfa1p is dependent on the central checkpoint regulator Mec1p. Analysis of the requirement for other checkpoint genes indicates that different mechanisms mediate radiation- and hydroxyurea-induced Rfa1p phosphorylation despite the common requirement for functional Mec1p. In addition, experiments with mutants defective in the Cdc13p telomere-binding protein indicate that ssDNA formation is an important signal for Rfa1p phosphorylation. Because Rfa1p contains the major ssDNA binding activity of the RPA heterotrimer and is required for DNA replication, repair and recombination, it is possible that phosphorylation of this subunit is directly involved in modulating RPA activity during the checkpoint response.