Genetic Recombination

Mammalian cells possess a large repertoire of repair processes that maintain the integrity of our genetic material. But some individuals have defects in DNA repair that can result in tumour formation: for example, defects in the repair of DNA double-strand breaks are responsible for some inheritable breast cancers. The research in our laboratory is focused on understanding how cells maintain chromosome stability to allow the faithful reproduction of the genome. In particular we want to understand how chromosomal breaks are repaired and how this process is important for tumour avoidance.

Our DNA is continually subjected to damage, either from endogenous sources such as reactive oxygen species that are produced as by-products of oxidative metabolism, from the breakdown of replication forks during normal cell growth, or by agents in the environment such as ionising radiation or carcinogenic chemicals. Fortunately, cells have evolved to cope with damage by employing elaborate and effective repair processes that are specialised to recognise certain lesions in DNA and to repair them.

DNA double-strand breaks represent one of the more dangerous forms of damage, as breaks can generate aberrant gene translocations. The consequence of such aberrant repair can be catastrophic to the cell and lead to cancer. One major pathway of double-strand break repair involves the enzymes of genetic recombination. Although genetic recombination normally occurs in germ-line cells at meiosis, where it provides a mechanism for the exchange and reassortment of genetic information, it also plays a critically important role in somatic cells for the repair of damaged or broken chromosomes. Our interest in the contribution of genetic recombination to the repair of DNA double-strand breaks stems from observations indicating that cell lines derived from individuals predisposed to breast cancer through mutations in BRCA2 exhibit a genome instability phenotype characteristic of a recombination/repair defect.

Recombinational repair and breast cancer
The importance of gaining a thorough understanding of homologous recombination is highlighted by observations indicating that individuals with mutations in BRCA2 have an extremely high probability (70% during their lifetime) of developing breast or ovarian cancers. The process of homologous recombination (HR) requires a number of proteins including RAD51, RAD52, RAD54, the RAD51 'paralogs' (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3), BRCA2 and RP-A. Many of these proteins have been purified in this laboratory, and we use biochemical, cytological and molecular biological approaches to understand how they function within the cell to repair DNA breaks.

Of particular importance for HR is RAD51, a protein that catalyses the key reactions required for DNA pairing and strand exchange. In response to DNA damage RAD51 localises to distinct sub-nuclear assemblies (foci) where the repair reactions take place. The localisation of RAD51 to repair foci is dependent upon the breast cancer-associated tumour suppressor BRCA2, with which RAD51 interacts. Our recent studies have provided a more detailed understanding of how BRCA2 controls RAD51 activity throughout the cell cycle and in response to DNA damage. We found that RAD51 interacts with the BRC regions of BRCA2 that map within exon 11, and at an unrelated site close to the C-terminus of BRCA2. The C-terminal RAD51- interaction domain of BRCA2 contains a unique site (S3291) that is phosphorylated by cyclin-dependent kinases (CDKs) and serves as a molecular switch that regulates interaction with RAD51. Using electron microscopy we have been successful in visualising the interaction of the C-terminal region of BRCA2 with active RAD51-DNA complexes (Esashi et al., Nature Struct Mol Biol 2007; 434: 598). We found that this region of BRCA2 interacts with the RAD51- DNA filament and helps improve filament stability. These results shed new light on the interplay between the BRCA2 tumour suppressor and RAD51 recombinase and are important to understand why C-terminal deletions of BRCA2 lead to cancer predisposition.

We also analysed the role of BRCA2 in regulating homologous recombination during meiosis. In addition to its known interactions with RAD51, we found that BRCA2 interacts with DMC1 protein, a meiosis-specific recombinase that is a homolog of RAD51 (Thorslund et al., EMBO J 2007; 26: 2915). The sites of interaction between BRCA2 and DMC1 were mapped in detail, revealing that they occurred at motifs distinct from those involved in RAD51 interactions. The ability of BRCA2 to interact with both RAD51 and DMC1 leads us to suggest that BRCA2 is a universal regulator of recombinase activities.

Crosslink repair and Fanconi anaemia
Fanconi anaemia (FA) is a rare autosomal disorder characterised by congenital abnormalities, bone marrow failure and increased incidence of cancer. Cells derived from individuals with FA exhibit a chromosome instability phenotype and are hypersensitive to agents that form DNA crosslinks. These cell lines can be classified into 12 complementation groups, and the genes responsible have now been identified. One of these genes is FANCM, which encodes a putative DNA helicase that is thought to be involved in the recognition of crosslinks as they impede the progress of the DNA replication apparatus. We recently discovered a novel protein, known as FAAP24, which interacts with FANCM to form a stable heterodimer (Ciccia et al., Mol Cell 2007; 25: 331). We found that FANCM/FAAP24 interacts with the Fanconi anaemia core complex, and suggested that it may play an important role in recruiting other FA proteins to sites of repair.

Defective DNA repair and neurological disorders
Defects in some DNA repair processes are associated with neurological disorders (Rass et al., Cell 2007; 130: 991). For example, individuals with Ataxia Telangiectasia exhibit progressively impaired balance and speech, cerebellar atrophy, and radiosensitivity, due to defects in a protein kinase that is required for the DNA damage response. Many also develop cancers, most frequently acute lymphocytic leukaemia and lymphoma. We have become interested in a similar, but genetically distinct, disorder known as Ataxia with Oculomotor Apraxia1 (AOA1), which is due to defects in a protein known as Aprataxin. When we characterised the biochemical properties of Aprataxin we found that it acts as a proofreader for DNA ligases (Ahel et al., Nature 2006; 443: 713, and Rass et al., J Biol Chem 2007; 282: 9469). Since neuronal tissues are subjected to elevated levels of oxidative stress that results in DNA damage, our observations suggest that the neurological problems associated with AOA1 are caused by the progressive accumulation of persistent nicks that cannot be repaired when Aprataxin is inactive. Importantly, these new data appear to define both the causative lesion associated with AOA1 and the molecular defects associated with this crippling neurological disease.

For a list of refereed research papers, see Publications (in navigation on left).