Meiotic recombination 11 (MRE11) is part of the MRE11-RAD50-Nbs1 (MRN) complex and is known to be involved in the resection of DNA strand ends in double strand break (DSB) repair as well as being involved in DNA replication. However, this latter topic is currently under researched. What is known about this is that the MRN complex has an impact on the restarting of replication forks which have been blocked or interrupted. The exact role that MRN and MRE11 have on replication fork restart however is not yet understood.

Nucleoside analogues have been widely used in treating various cancers and work by interfering with DNA replication by inhibiting nucleoside metabolism as well as acting as DNA replication chain terminators. This prevents the cells from restarting the replication process. The nucleoside analogues Gemcitabine and Cytarabine have been shown to be integrated into genomic DNA during replication and acting as chain terminators, thus proving useful anti-cancer treatments. The removal of Gemcitabine has been shown to be affected by MRE11 nuclease activity, with a lack of removal displayed when inhibiting MRE11 nuclease activity. With cells unlikely to come across these nucleoside analogues in nature, it is possible that this role of MRE11 has evolved as a reaction to chain terminating altered nucleosides already present in the nucleoside/nucleotide pool. One such nucleoside with chain terminating ability, once integrated into DNA, is 8-oxo-2’-deoxyguanosine triphosphate (8-oxodGTP). 8-oxo2’-deoxyguanosine (8-oxodG) is one of the most common biomarkers for oxidative stress and can be mis-incorporated into DNA in its tri phosphorylated form, resulting in chain termination.

Within this project, experiments were performed in order to test the contributions of MRE11 nuclease activity in the resistance towards 8-oxodG in various strains of DT40 chicken cells. These experiments provided interesting results, suggesting that MRE11 exonuclease activity is involved in the resistance of cells towards 8-oxodG treatment. These results also suggested that polymerase epsilon nuclease activity does not contribute to the resistance of 8-oxodG in these cell lines.

Secondly, DNA purification experiments were performed in order to test whether 8-oxodG was integrated into genomic DNA, deducing whether the cytotoxicity seen towards the cells was caused by 8-oxodG treatment. This was tested using mass spectrometry analysis. Mass spectrometry is a common technique used to measure the amount of 8-oxodGTP that can be integrated into DNA. However, when preparing samples for mass spectrometry analysis, problems can occur. The artefactual oxidation of dG into 8-oxodG during sample preparation is a major problem, resulting in an overestimation in the amount of 8-oxodG seen upon mass spectrometry analysis due to raised background levels of this oxidised nucleotide. The results from our experiments confirmed that artefactual oxidation of 8-oxodG was indeed likely to be a problem when using this analytical technique. This led to further experiments, with the aim of accurately measuring the amount of 8-oxodG that was incorporated into genomic DNA. However, high 8-oxodG levels were detected following our techniques to reduce artefactual oxidation, so a definitive conclusion could not be determined.

The synthesis of an isotopically labelled variant of 8-oxodG was also attempted within this project, given the success that isotopes can have when differentiating between compounds using mass spectrometry analysis. This would allow for a clear differentiation between integrated and artefactually oxidised 8-oxodG. This synthesis provided difficulties with producing a high enough product yield for detection using mass-spectrometry analysis, so it remains unclear whether this method could be a viable option for differentiating between 8-oxodG variants in future work.