Ubiquitin-mediated proteolytic degradation of the small subunit of DNA polymerase delta: mechanism, regulation and impact on genome stability

DNA polymerase delta functions in lagging strand DNA replication (Okazaki fragment synthesis) and DNA repair in eukaryotic cells, and comprises four subunits: PolD1 (p125), PolD2 (p50), PolD3 (p68) and PolD4 (p12). Work in mammalian cells has implicated degradation of PolD4 (p12) in the cellular response to DNA damage induced by UV light, although exactly how this occurs remains to be elucidated.

The proposed project will see the student investigate in greater detail the mechanism, regulation and impact of PolD4 (p12) degradation on chromosomal DNA replication and repair using the genetically tractable fission yeast Schizosaccharomyces pombe as a model system. We have shown that, as in human cells, the fission yeast PolD4 protein (known as Cdm1) is degraded by the CRL4 (Cullin-RING ubiquitin protein ligase 4/Cdt2)-mediated proteolysis pathway, but how and why exactly this occurs is unclear. To investigate this, the student will map the sequences on Cdm1 that trigger its degradation and investigate the consequences of degrading Cdm1 (or preventing its degradation) on cell cycle progression and genome stability. In addition, the student will purify the PolD4 protein with a view to characterising in detail its interactions with the other subunits of Pol delta and to determining its three-dimensional structure (by NMR). Through this work, the student will build up a detailed picture of the part played by PolD4 degradation in the maintenance of eukaryotic genome integrity.

The student will join a well-equipped lab with diverse interests in cell cycle control and chromosomal DNA replication and repair in yeast, archaea and trypanosomes (see http://biology.st-andrews.ac.uk/macneill for details), located in the recently opened Biomedical Sciences Research Complex (www.st-andrews.ac.uk/bsrc). The successful applicant should have a specific interest in the application of model systems to tackle questions of protein structure and function in eukaryotic cells. He/she will receive extensive training in the molecular genetic, molecular biological and biochemical techniques required to bring the project to a successful conclusion and will have opportunity to attend national and international meetings to present his/her data and to collaborate with groups elsewhere in St Andrews, the UK and abroad. Training will be further enhanced by participation in GRADskills, an extensive and award-winning generic skills development programme for early stage researchers. Informal enquiries can be directed to Dr Stuart MacNeill ([Email Address Removed]).