Cell cycle regulation: the role of replication clamps and interacting proteins

DNA replication is executed by enzymes called polymerases. The polymerases are tethered to the DNA template by binding to so-called clamps, which are doughtnut-shaped protein complexes encircling the DNA. The clamps are being loaded onto the DNA by clamp loaders, another protein complex. All organisms have clamps and clamp loaders, and they have similar structures and functions. It is known that the clamps bind polymerases and other proteins associated with the replciation fork. The object of the project is to identify and characterise proteins interacting with the clamps. We will investigate clamps and clamp loaders both yeast cells and to some extent in bacterial cells. At present, some interacting proteins are known and some are presumed. In the yeast system we will mainly study an alternative clamp/clamp loader pair which is acting in handling damage to DNA. The functions of this clamp/clamp loader are not at all clear, but they are absolutely required for cellular response to DNA damage and to halt t he cell cycle progression until the DNA is repaired. It may also be involved in repairing DNA damage. Many proteins have been found to interact with the regular clamp (called PCNA), and we presume that many proteins will likewise interact with the alterna tive clamp. We will put a molecular tag onto the proteins in question (the clamp), which allows them to bind strongly to a solid matrix. Proteins binding to the clamp will therefore also bind to the matrix, and we can investigate the nature of these prote ins.