DNA repair mechanisms are essential not only to allow cells to recover from radiation or damaging chemicals, but also to maintain the stability and integrity of the genetic material during normal growth and development. Defects in repair genes usually have devastating consequences, conferring human diseases such as xeroderma pigmentosum, ataxia telangiectasia or nijmegen breakage syndrome. In addition, errors introduced into DNA as part of repair in normal individuals represent a major mechanism for malignant transformation. Hence, it is important to understand the molecular mechanisms cells use to maintain the integrity of their genome, and to do this, it is necessary to understand the role of several different repair processes. The RAD genes of budding yeast (Saccharomyces cerevisiae) have become a key model system for studying DNA repair mechanisms, and have led the way to identifying many human repair genes. This proposal seeks to continue this research into yeast RAD genes using exciting new methods that have not yet been exploited. The cell's ability to resolve double strand breaks (DSBs) in DNA is crucial in many aspects of repair and genome maintenance, and is a focus here. A method has been developed to measure DSBs and their repair more accurately than previously, and this permits a new characterization of RAD gene pathways. The method makes use of a ring chromosome in heterozygous condition with a linear homologue. The once-broken circles form a unique linearized-circle band on pulsed field gels, whose intensity in relation to the unbroken linear- homologue molecules defines the mean number of breaks per molecule. This method will be used to determine how many breaks per cell can be tolerated in different genotypes, and to measure the efficiency of repair. Despite the importance of DSBs, it is not clear that they are responsible for most lethality in wild-type, and this will be tested by measuring how many DSBs remain after repair in wild-type at physiological doses. Epistasis relationships amongst X-ray sensitive mutants will be determined in detail using double and multiple mutant analysis. This approach was very successful in determining pathways of ultraviolet radiation repair, but has not been used with the same rigor for X-ray sensitive mutants. It is important in regard to recent findings that excision repair and Ku-complex mediated end-joining repair also contribute to X- ray resistance in yeast. In a related aim, the order of gene function for RAD5O and RAD54 will be tested using different switching regimes with double mutants containing conditional alleles with differing permissive conditions.
| Game, John C; Williamson, Marsha S; Spicakova, Tatiana et al. (2006) The RAD6/BRE1 histone modification pathway in Saccharomyces confers radiation resistance through a RAD51-dependent process that is independent of RAD18. Genetics 173:1951-68 |
| Game, John C; Williamson, Marsha S; Baccari, Clelia (2005) X-ray survival characteristics and genetic analysis for nine Saccharomyces deletion mutants that show altered radiation sensitivity. Genetics 169:51-63 |
| Game, John C; Birrell, Geoff W; Brown, James A et al. (2003) Use of a genome-wide approach to identify new genes that control resistance of Saccharomyces cerevisiae to ionizing radiation. Radiat Res 160:14-24 |
| Game, John C (2002) New genome-wide methods bring more power to yeast as a model organism. Trends Pharmacol Sci 23:445-7 |
