DNA double strand breaks (DSBs) are one of the most deleterious lesions to a cell and can lead to cell death or carcinogenesis. DSBs can be caused by exogenous agents such as ionising radiation, or endogenous agents such as oxygen radicals. DSBs can be repaired by two different cellular pathways: homologous recombination (HR) and the more error-prone non-homologous end-joining (NHEJ). While HR uses a homologous DNA sequence as a template for repair, NHEJ directly ligates DSB ends.

Repair by NHEJ is characterised by the presence of the heterodimeric protein complex Ku 70/80 ,which encircles the DNA at the break site. If the DNA requires processing, prior to ligation, this is carried out by a variety of polymerases, nucleases and other enzymes that are recruited in a Ku-dependent manner. Ligation of the break is performed by DNA ligase IV (Lig4), which is well conserved throughout eukaryotes. In vertebrates , the ligase complex also contains two other proteins. XRCC4, which binds strongly to Lig4 conferring protein stability, and XLF. The exact  functional role of these factors in the DSB repair process remains unclear.

While mammalian cells use NHEJ as the preferred DSB repair mechanism, yeast  (e.g. S.cerevisiae and S.pombe) repair DSBs predominantly by HR in their vegetative state. Schizosaccharomyces pombe has been used extensively as a model organism to study cellular processes, including cell cycle events. In order to gain further insight into the importance of NHEJ, we are using Schizosacchromyces pombe as a model to study different aspects of the NHEJ pathway.

Our work has led to the discovery of Xlf1, a homologue of mammalian XLF, which is a novel factor required for S.pombe NHEJ. Fission yeast xlf1 is required for the religation of linearised plasmids by NHEJ and interacts with DNA ligase IV. In addition, xlf1 is required for the survival of DSB under certain physiological conditions.

We are also interested in the regulation of  double-strand break repair pathway choice and the cell cycle dependent aspects of this process. As NHEJ can be utilised throughout all stages of the cell cycle, its down regulation is likely to be key during  post-replication stages when HR will be favoured by the cell. Using cell cycle deficient mutants and nutrient depletion, it is possible to study the different cycle phases of S.pombe and compare and contrast factors that are directly influencing HR and NHEJ pathway selection.

Co-workers
Pierre Hentges
Helen Waller

References
Hentges, P., Ahnesorg, P., Pitcher, R.S., Bruce, C.K., Kysela, B., Green, A.J., Bianchi, J. Wilson, T.E., Jackson S.P. & Doherty, A.J. (2006)
Evolutionary and Functional Conservation of the DNA Non homologous End‑joining Protein, XLF/Cernunnos.
J. Biol. Chem. 281, 37517–37526.