Formation, identification, and repair of gamma-radiation-induced DNA-protein crosslinks in mammalian cells

DNA-protein crosslinks (DPCs) are induced by a variety of commonly encountered agents, including metals, aldehydes and radiation, as well as chemotherapeutic drugs. There is tremendous variability in the literature with respect to our understanding of the induction, stability, and proposed repair pathways for DPCs induced by various agents. Different crosslink chemistries are likely to affect the impact, stability, and repair of these lesions. As well, a number of different methods have been used for DPC measurements, which has probably contributed to the variability seen in DPC analyses.; Ionizing radiation (IR) is an important environmental risk factor for various cancers and also a major therapeutic agent for cancer treatment. Exposure of mammalian cells to IR induces several types of damage to DNA, including double and single strand breaks, base and sugar damage, as well as interstrand crosslinks (ICLs) and DPCs. The biological consequences of DPC-inducing agents, such as IR, include increased mutagenesis, chromosomal aberrations and cytotoxicity, but the contribution of DPCs to these endpoints has not been well characterized. The determination of the consequences of DPC induction will require an assessment of which proteins become crosslinked to DNA and the stability of these linkages.; This work describes the development and validation of novel methodology for the isolation of DPCs from mammalian cells using chaotropic agents to isolate genomic DNA and stringently remove non-crosslinked proteins followed by nuclease digestion to release covalently crosslinked proteins. This method generates high quality protein samples in sufficient quantities for analysis by mass spectrometry.; Using our DPC isolation method in combination with mass spectrometry, we have identified 29 proteins belonging to numerous functional categories that can become crosslinked to DNA by gamma-radiation under aerated and/or hypoxic conditions. Several aspects of DPC induction were examined by staining 1-D SDS-PAGE gels with SYPRO Tangerine followed by analysis using fluorescence imaging. DPCs were induced linearly with IR dose at low doses, but appeared to reach a plateau with higher doses. There was no dramatic influence of oxygen on total IR-induced DPCs observed. DPC removal was observed to be limited in repair-deficient cell lines. Measurements of the extent and timecourse of protein crosslinking will ultimately contribute to the understanding of the effect of DPCs.