Cortez Lab

The ATR and ATM kinases are at the apex of the DNA damage response signaling pathway. These large protein kinases are members of an atypical protein kinase family that includes DNA protein kinase (DNA-PK) and target of rapamycin (TOR). Mutations in ATR cause the rare disease Seckel syndrome and mutations in ATM cause the childhood neurodegenerative and cancer-predisposition disorder ataxia-telangiectasia. We have an ongoing project to understand the regulation of these kinases in response to DNA damage. ATM is primarily activated in response to double strand breaks while ATR is activated by agents that cause replication stress such as ultraviolet radiation and the chemotherapeutic agents cisplatinum, hydroxyurea, and etoposide. Activation of these kinases involves at least two steps. The kinases are recruited to the sites of DNA damage and then biochemical changes in the proteins allow them to phosphorylate substrates. Key substrates include p53, Nbs1, Mre11, Chk2, and Chk1. Many of these substrates are themselves encoded by tumor suppressor genes. p53, for example, may be the most widely mutated genes in human cancer. We are using biochemical, genetic, and cell biological techniques to determine how these kinases are initially activated in response to genotoxic stress.

Localization is a key step in checkpoint kinase activation. In the past couple of years we have made significant progress in understanding how the ATR kinase recognizes DNA damage. The ATR kinase has a subunit called ATRIP that is required for its localization. ATRIP has an N-terminal RPA binding domain. RPA is a single-stranded DNA (ssDNA) binding protein required for most DNA metabolism including replication, repair, and recombination. The interaction between ATRIP and RPA helps pull the ATR-ATRIP complex to regions in the genome that have ssDNA. Since ssDNA accumulates at DNA lesions due to both DNA metabolism and pausing of DNA polymerases, RPA-ssDNA complexes can act as a marker for problems in the genome. Our genetic and biochemical approaches have started to reveal the molecular details of the ATRIP-RPA interaction and structural studies are beginning to provide us the ability to visualize how ATR recognizes DNA damage.



 

Localization is only the first step in activating the ATR checkpoint kinase. We know that many other proteins play critical roles in promoting ATR-dependent phosphorylation of substrates. TopBP1 binds and activates the ATR kinase in vitro. The mechanism by which this occurs is unclear. A group of eight proteins including Rad17 and Rad9 are critical for ATR activation as well. These proteins may also serve as sensors of ssDNA-RPA. Perhaps they help bring TopBP1 into contact with ATR to promote ATR activation. Some ATR substrates also have specific adaptor proteins that are required for their phosphorylation. For example, Chk1 phosphorylation and activation by ATR requires claspin. Finally, many of these proteins are substrates of ATR. A variety of projects are ongoing in the lab to sort out how all of these proteins function to promote ATR signaling.