The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast

Abstract

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.

Checkpoint-mediated control of replisome–fork association and signalling in response to replication pausing

“…These aberrant intermediates include hemireplicated and gapped molecules likely resulting from lagging strand defects (Lopes et al, 2001;Sogo et al, 2002). This hypothesis is supported by the findings that the lagging strand replication apparatus (the DNA polymerase alpha-primase complex (pol-prim) and replication factor A (RFA)) is regulated in response to checkpoint activation (Brush et al, 1996;Pellicioli et al, 1999;Brush and Kelly, 2000), and that certain pol-prim and RFA mutants exhibit allele-specific checkpoint defects (Longhese et al, 1996;Marini et al, 1997).…”

ATM: A mediator of multiple responses to genotoxic stress

“…In this organism, the phosphorylation of RPA2 requires functional MEC1, an ATM homolog (Brush et al, 1996). In mammalian cells, both DNA-PK and ATM were implicated in DNA-damage induced phosphorylation of RPA2.…”

The YeastTEL1Gene Partially Substitutes for HumanATMin Suppressing Hyperrecombination, Radiation-Induced Apoptosis and Telomere Shortening in A-T Cells

“…Since Tel1p shares 45% amino acid identity in the kinase domain with ATM, and since TEL1 controls damage-induced phosphorylation of Rfa2p, Rad53p, and Rad9p in yeast (Brush et al, 1996;Sugimoto et al, 1997;Emili, 1998;Vialard et al, 1998), we asked whether Tel1p may modulate p53 in transfected A-T cells and, thus, contribute to the complementation of cellular phenotypes. Although p53 is bound and, thus, inactivated by a large T-antigen in SV40-transformed cells, a retained regulatory mechanism of p53 activity in SV40-transformed human cells has been suggested (Jung et al, 1997).…”

“…Both are required for phosphorylation of downstream proteins following DNA damage. IR-induced phosphorylation of p53, c-abl, and Rad51 in human cells is ATM dependent (Baskaran et al, 1997;Shafman et al, 1997;Banin et al, 1998;Canman et al, 1998;Kharbanda et al, 1998;Yuan et al, 1998), and TEL1 controls damage-induced phosphorylation of Rfa2p, Rad53p, and Rad9p in yeast (Brush et al, 1996;Sugimoto et al, 1997;Emili, 1998;Vialard et al, 1998). TEL1-deficient yeast cells express an A-T like genome instability that includes chromosome loss, hyperrecombination, and telomere shortening (Lustig and Petes, 1986;Greenwell et al, 1995).…”