Upon photoactivation, cross-linking agents like psoralens can alkylate both strands of DNA, generating interstrand cross-links (ICLs) that are absolute blocks to replication and transcription. Although psoralens have been widely applied as anticancer drugs in chemotherapy regimens, our understanding of the repair mechanism of ICLs in cells remains incomplete. Here we reported a method that encompassed nuclease P1 digestion and LC-MS/MS with the isotope-dilution technique to monitor the repair of 8-methoxypsoralen (8-MOP) photo-induced ICLs in vivo. By using CD3-8-MOP-housing ICLs as internal standard, 8-MOP ICLs could be quantified at the level of 1 lesion per 106 unmodified nucleotides in mammalian cells. Additionally, our results revealed that ICL is repaired more efficiently in repair-competent cells than in cells that are defective in nucleotide excision repair (i.e., XPA- or ERCC1-deficient). We are now in the process of assessing how deficiency in other repair factors compromises the repair of 8-MOP ICLs in cells.

Mass spectrometry-based approach for assessing the repair of DNA interstrand cross-links introduced by 8-methoxypsoralen in mammalian cells

244th National Meeting (2012)

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