DNA Repair 3 (2004) 901–908 Review Regulation of DNA replication by ATR: signaling in response to DNA intermediates David Shechter a,b , Vincenzo Costanzo b , Jean Gautier b,c,∗ a Integrated Program in Cellular, Molecular, and Biophysical Studies, Columbia University College of Physicians and Surgeons, 701 W. 168th Street, New York, NY 10032, USA b Department of Genetics and Development, Hammer Health Sciences Center, Room 1620, Columbia University College of Physicians and Surgeons, 701 W. 168th Street, New York, NY 10032, USA c Hammer Health Sciences Center, Room 1602A, Columbia University College of Physicians and Surgeons, 701 W. 168th Street, New York, NY 10032, USA Available online 9 April 2004 Abstract The nuclear protein kinase ATR controls S-phase progression in response to DNA damage and replication fork stalling, including damage caused by ultraviolet irradiation, hyperoxia, and replication inhibitors like aphidicolin and hydroxyurea. ATR activation and substrate specificity require the presence of adapter and mediator molecules, ultimately resulting in the downstream inhibition of the S-phase kinases that function to initiate DNA replication at origins of replication. The data reviewed strongly support the hypothesis that ATR is activated in response to persistent RPA-bound single-stranded DNA, a common intermediate of unstressed and damaged DNA replication and metabolism. © 2004 Elsevier B.V. All rights reserved. Keywords: ATR; DNA replication; Checkpoint; Single-stranded DNA; RPA 1. Introduction ATR (ATM- and rad3-related) is a large and essential checkpoint protein kinase in metazoans. ATR is similar in function to the S. cerevisiae gene product Mec1 and critical for the DNA damage response that halts the pro- gression of the cell cycle, in particular S-phase, upon UV irradiation, hyperoxia, DNA alkylation, and treatment with drugs like aphidicolin, a DNA polymerase inhibitor. Ataxia-telangiectasia-mutated gene product (ATM), in contrast, serves to modulate the DNA damage response upon sensing DNA double-strand breaks. We argue that ATR is responsible for checkpoint signaling in response to a transient intermediate of both normal and stressed metabolism: single-stranded DNA. This ability of ATR to regulate the cell cycle in response to such a common DNA structure makes it a crucial player in the life of a cell. ∗ Corresponding author. Tel.: +1-212-305-9573 (lab)/1-212-305-9586 (office); fax: +1-212-923-2090. E-mail addresses: ds453@columbia.edu (D. Shechter), vc151@columbia.edu (V. Costanzo), jg130@columbia.edu (J. Gautier). Duplication of an intact, complete genome is the ulti- mate goal of progression through the cell cycle. Eukaryotic cells rely on cell-cycle regulation to ensure proper timing and completion of DNA replication originating from multi- ple origins. Importantly, the asynchronous nature of origin activation as S-phase progresses provides a potential mech- anism to halt further replication, in the presence of DNA damage or any genotoxic stimuli, as unfired origins can be downregulated. Checkpoint signaling networks are biological transistors that read and integrate damage or replication-state inputs and decide an outcome: permit progression or halt the cell cycle to allow repair or apoptosis. In the context of S-phase, the checkpoint pathways are responsible for sensing aber- rant DNA structures or replicative stress on the genome and preventing progression through the cell cycle that would re- sult in mutations or loss of genetic information due to DNA damage. DNA-damage checkpoints are triggered by an activating signal, likely from the DNA template itself. These dam- age signals result from both extrinsic and intrinsic events. Genotoxic stresses, like ionizing radiation (IR), ultraviolet light (UV), nucleotide depletion, and oxidative products can 1568-7864/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2004.03.020