The DNA damage response is important for maintaining genomic integrity following introduction of double-strand breaks (DSB) since illegitimate or incorrect repair of a DSB could promote malignant transformation. Mismatch-repair (MMR) deficient tumour cell lines are acutely sensitive to thymidine treatment and fail to activate homologous recombination (HR) repair following a DSB (Mohindra et al., 2002). Therefore, it was hypothesised that loss of HR repair may occur as a downstream event in tumours already deficient in MMR. The primary aim was to determine whether there were somatic mutations in candidate HR genes that were associated with colorectal cancer development and to examine the phenotypic consequences of such mutations. Analysis of the candidate HR genes, XRCC2, XRCC3 and Mus81 in a random collection of primary colorectal cancers and in a specific MMR-deficient tumour population revealed no tumour-specific mutations. Analysis of the MREI J, NBSI and Rad5O genes revealed a frameshift mutation in an intronic T11 tract of MRE11 that gave rise to alternative splicing of the gene. This mutation was present in >85% of MSI+ colorectal tumours suggesting that alteration of MRE11 occurs at a high frequency in tumours already deficient in MMR. The phenotypic consequences of MRE11 dysfunction were investigated by expressing splice variants of MRE 11 in MMR-proficient human cells. Cells expressing an MRE 11 variant with a compromised nuclease domain, failed to activate wild-type MRE11 following DNA damage, were dramatically more sensitive to thymidine and failed to activate HR repair following thymidine treatment. The novel results obtained here suggest that a functional MRE 11 is essential for the HR repair-mediated rescue of DNA replication forks impaired by thymidine. Furthermore, it was speculated that a functional MRE11 may be essential to resolve the recombinogenic substrate specifically produced by thymidine. Given that this DNA damage response pathway appears to be disrupted in >85% of MSI+ colorectal tumours, these finding have important implications for treatment strategies directed against this subset of tumours.