The cells of an organism must be able to accurately duplicate and pass on its genetic material to its daughter cells in order to exhibit proper functioning and survival. However, the genomic stability of a cell is constantly being pressured by extrinsic DNA damaging agents as well as intrinsic stresses caused by regular metabolic processes. Eukaryotic cells have developed a complex system of regulatory mechanisms called cell cycle checkpoints to ensure that certain cellular processes are completed before downstream cell cycle events can occur by delaying or arresting cell cycle progression. The many proteins and processes involved in the checkpoint response often overlap and function interdependently with the mechanisms of DNA damage repair, and apoptosis. The cell cycle checkpoints regulate entry of cells into S-phase, progression through S-phase, and entry into and exit out of mitosis in response to DNA damage. Due to the importance of cell cycle checkpoints in maintaining genetic stability, defects in this system can contribute to diseases such as cancer.
The accumulation of genetic mutations in cells causes the activation of protooncogenes and the inhibition of tumor suppressor genes that can lead to cancer formation. Defects in the mechanisms of DNA repair and cell cycle checkpoints may allow cells to continue dividing in the presence of DNA damage. One of the most commonly mutated genes in human cancer is p53, which has a number of roles involved in DNA repair, apoptosis and cell cycle checkpoint control. Other cell cycle checkpoint proteins implicated in various cancers include Chk1, Chk2, BRCA1, and ATM. Recently, cell cycle checkpoint proteins have also been increasingly studied as targets for therapeutic cancer drugs. Although the loss of checkpoints can lead to uncontrolled cell proliferation, it can also lead to cell death, which can be desirable for the treatment of cancer. Many cancer cells have intact G2/M checkpoints, while they have defects in the G1/S checkpoint. Thus, the targeting of G2/M checkpoint proteins such as Chk1 have been of keen interest in recent years for the study of cancer therapeutics. Further insight into the mechanisms and specific protein interactions of the various checkpoint signaling pathways in the checkpoint response to DNA damage will be valuable for the study and treatment of human cancers.
The proteins involved in the DNA damage cell cycle checkpoints are generally grouped into four categories; sensors, transducers, mediators, and effectors. However, these are loosely defined categories for conceptual purposes and many proteins can be thought of as belonging to more than one group. Also, although each DNA damage checkpoint pathway is unique, they may share many of the same proteins in their signaling cascade.
Cell cycle checkpoint sensors are the proteins/protein-complexes that recognize the sites of DNA damage or replication stress and initiate the checkpoint response. One important sensor complex in the DNA damage checkpoint response is made up of the Rad17-RFC (Replication Factor-C) complex and the 9-1-1 complex. The related ATM (Ataxia Telangiectasia Mutated) and ATR (Ataxia telangiectasia and Rad3 related) kinases also act as sensors, responding to different types of DNA damage. Although these sensors all respond to DNA damage, they are thought to be loaded to sites of damage independently of one another.
The mediator group of cell cycle checkpoint proteins is recruited to sites of DNA damage by the sensors and may interact with both ATM/ATR and downstream transducers to relay specific checkpoint signals. Mediators include Topoisomerase Binding Protein 1 (TopBP1), Claspin, Mediator of DNA Damage Checkpoint Protein 1 (MDC1), and p53 Binding Protein 1 (53BP1). In the DNA damage checkpoint response, the main transducers are the Chk1 and Chk2 kinases. Although there is overlap between the pathways, generally the damage signal sensed by ATR is transduced by Chk1, while that of ATM is transduced by Chk2. These transducer kinases ultimately relay the damage signal to proteins such as the Cdc25 phosphatases and p53 that directly target the effector proteins responsible for cell cycle transition, which are primarily cyclindependent kinases (Cdks).
Checkpoint activation by DNA damage
Exposure to different types of genotoxic agents, and even regular metabolic processes such as replication can lead to the formation of DNA structural abnormalities or intermediates. These include stalled replication forks, double strand breaks (DSBs), pyrimidine dimers, and other structural changes of DNA that activate the cell cycle checkpoint response. As noted earlier, DSBs are associated primarily with the activation of ATM and is recognized as one of the most dangerous forms of DNA damage. Ionizing radiation (IR) sources can easily induce many types of DNA lesions, including DSBs and is used commonly in the laboratory for this purpose. IR is able to ionize molecules with which it collides, causing damage directly to DNA or indirectly through the production of reactive oxygen species. Another common experimental compound for checkpoint activation is hydroxyurea (HU). HU works by blocking DNA replication through the inhibition of the enzyme ribonucleotide reductase (RNR), which is responsible for the conversion of ribonucleotides into deoxyribonucleotides. Nucleotide depletion leads to stalled replication forks, creating stretches of ssDNA that primarily activates the ATR-mediated checkpoint response.