An efficient DNA damage response is critical for maintaining the integrity
of the mammalian genome, and ensuring the accurate transfer of genetic
information between generations. Of particular biological relevance are
DNA double strand breaks (DSB), which if repaired incorrectly may
contribute to carcinogenesis.
Review of contemporary literature has led to the identification of protein
interactions and transcriptional events, tightly associated with the
mammalian DSB response. Characteristics of selected events have been
manipulated, with the notion of developing a reporter system that offers a
sensitive and rapid method of detecting DSB in living mammalian cell
models. Work presented here provides a quantitative evaluation of DSB
generation in various mammalian cell lines, following chemical and
irradiation treatment, and highlights the limitations of currently used
markers.
A series of recombinant proteins comprising peptide interacting domains,
which exhibit altered spatio-temporal dynamics in relation with each other
following DSB induction, are proposed as potential reporters of damage
in mammalian cells. Novel gene constructs have been engineered that
encode these peptide interacting domains, sandwiched between
fluorescence-resonance-energy transfer (FRET) capable proteins. DSB
specific events are predicted to induce peptide interactions that may be
tracked in real time, by monitoring alterations in the fluorescent properties
of such a recombinant protein.
In an alternative approach, the transcriptional up-regulation of RAD52
mRNA following DSB induction was extended to whole cells. Optimisation
of a fluorescent molecular beacon probe complementary to mammalian
RAD52 mRNA is described, and data obtained in mammalian cells
following DSB induction supports the notion that RAD52 is actively
transcribed as part of the DSB response.