Cell cycle checkpoints and DNA repair mechanisms help to maintain a cell's genomic integrity. Inactivation of genes required for these processes has been linked to syndromes causing predisposition to cancer. The DNA damage checkpoint involves the detection of damaged DNA and the generation of a signal that delays the cell cycle in order to allow time for the damaged DNA to be repaired. Proteins required for the DNA damage checkpoint have been identified primarily through genetic screens in yeast, and the human homologs have recently been cloned. Despite these momentous advances, the DNA damage checkpoint remains biochemically ill-defined. In particular, it is not known how the DNA damage is sensed. Six proteins in fission yeast have been implicated in sensing damage and activating the checkpoint kinases. It is not known whether these proteins directly interact with the DNA lesion or indirectly by interacting with repair complexes, or stalled transcription or replication complexes. One of these proteins, Rad17, has sequence homology to replication factor C (RFC) subunits, and has been shown to associate with RFC subunits in yeast. Rad17 hydrolyzes ATP in the process of loading PCNA onto DNA during replication and repair. RAD17 may perform a similar function to load checkpoint proteins onto DNA. The main goal of this proposal is to determine how hRad17 functions in the DNA damage checkpoint. This will be accomplished by: 1) determining if hRad17 binds and/or hydrolyzes ATP; 2) identifying proteins that interact with hRad17; and 3) determining if hRad17 associates with specific DNA structures, repair complexes, or repair intermediates. The results from these experiments will further our understanding of the role of hRad17 in the DNA damage checkpoint and may provide the groundwork for new cancer treatments.
| Lindsey-Boltz, Laura A; Wauson, Eric M; Graves, Lee M et al. (2004) The human Rad9 checkpoint protein stimulates the carbamoyl phosphate synthetase activity of the multifunctional protein CAD. Nucleic Acids Res 32:4524-30 |
| Bermudez, Vladimir P; Lindsey-Boltz, Laura A; Cesare, Anthony J et al. (2003) Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro. Proc Natl Acad Sci U S A 100:1633-8 |
| Griffith, Jack D; Lindsey-Boltz, Laura A; Sancar, Aziz (2002) Structures of the human Rad17-replication factor C and checkpoint Rad 9-1-1 complexes visualized by glycerol spray/low voltage microscopy. J Biol Chem 277:15233-6 |
| Lindsey-Boltz, L A; Bermudez, V P; Hurwitz, J et al. (2001) Purification and characterization of human DNA damage checkpoint Rad complexes. Proc Natl Acad Sci U S A 98:11236-41 |
