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All organisms can suffer DNA double-strand breaks (DSBs), during meiosis, during DNA replication as elongating forks stall or collapse, or as a consequence of treatment with DNA damaging agents such as IR or MMC. If left unrepaired, the consequences for genomic stability would be disastrous, resulting in chromosomal deletions and translocations leading to apoptosis or carcinogenesis. It is becoming increasingly clear that tumour formation can be triggered by mutations in genes involved in the surveillance of the genome integrity such those for ATM, BRCA2 and RAD51. DSBs in human cells may be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). NHEJ, sometimes inaccurate, leads to the joining of DNA broken ends in a mechanism dependent on DNA-PK and the Ku70/Ku80 proteins. The HR pathway is an error-free way of repairing DSBs but it is slower than NHEJ. In eukaryotes, HR is initiated when the MRE11-RAD50-NBS1 (MRN) complex enters DNA at a DSB. MRE11 is a conserved protein with a N-terminal nuclease domain that has both 3'-to-5' exonuclease and endonuclease activites. The nuclease travels along the DNA leading to the resection of the DSB and the formation of single-stranded DNA tails. These are then acted upon by the RAD51 recombinase (a homolog of E. coli RecA) which forms a filament and catalyses invasion by the single-strand DNA of an intact sister chromatid to create the initial heteroduplex joint and a Holliday junction (HJ). This junction moves along the DNA (a process called branch migration) and is cleaved by a resolvase complex to complete the exchange of genetic information and DNA repair (Fig. 1E). NHEJ was thought to be the primary mechanism for the repair of DSBs in human cells. It now appears that HR and NHEJ mechanisms of repair are equally important but NHEJ plays a dominant role to repair DSBs during G1 to early S phase, while HR is used in late S to G2 when newly replicated chromosomes are available as homologous templates.
One individual out of three will develop a cancer during their lifetime. At present, radiotherapy using ionizing radiation is the most employed form of cancer therapy. This treatment shows severe cytotoxic effects on actively growing cancer cells by inducing DNA damage, mainly double-strand breaks. However, radiotherapy results in severe negative side effects, often creating new mutations which trigger other cancers. Radiotherapy is also ineffective in hypoxic tumors because the low oxygen tension of hypoxia does not result in the production of reactive oxygen species necessary for cytotoxicity. In contrast, the DNA damaging drug MMC is preferentially cytotoxic to hypoxic malignant cells because its requirement for bioreductive activation exploits the hypoxic environment, which favours reductive processes. Unfortunately, both IR and MMC treatments suffer from a major limitation: DNA repair often causes reduced sensitivity of cancer cells to the drugs. Hence, there is a pressing need to increase the effectiveness of these treatments. The objective of the biochemical and genetic experiments conducted in this laboratory aim to learn how MRN, the RAD51 paralogs and Fanconi anemia proteins repair lesions caused by anticancer agents such as IR and MMC and how they contribute to genome stability. Our goal is then to use the knowledge gained on repair processes and use it to sensitize cancer cells to anticancer agents.
Homologous recombination is also important during meiosis. Meiosis generates haploid gametes or four spores in yeast through a cell division that consists of one round of DNA replication followed by two cell divisions. These divisions are preceded by a unique meiotic prophase during which homologous chromosomes synapse and undergo recombination. At this stage, chromosomes from each parent recombine at frequencies that are 100-1000 fold higher than in vegetative cells leading to gametes with different genetic signatures. The consequences of meiotic recombination are gene rearrangements, genetic diversity and proper chromosome segregation. The successful segregation of homologs require the maintenance of physical connections between homologs, a role that is fullfilled by the sites of recombination (also known as chiasmata). This is of particular importance as failure to resolve chiasmata between homologs results in non-disjunction (non-separation of chromosomes) and aneuploidy (defined as a cell having a wrong number of chromosomes). Aneuploidy has profound clinical consequences since approximately one-third of all human miscarriage are aneuploid and aneuploidy is the leading genetic cause of developmental disabilities and mental retardation such as found in trisomy 21. A third project is to understand how homologous recombination contributes to aneuploidy and genetic diseases such as Down syndrome.