New Synthetic Analogs of Nitrogen Mustard DNA Interstrand Cross-Links and Their Use to Study Lesion Bypass by DNA Polymerases.

Nitrogen mustards are a widely used class of antitumor agents that exert their cytotoxic effects through the formation of DNA interstrand cross-links (ICLs). Despite being among the first antitumor agents used, the biological responses to NM ICLs remain only partially understood. We have previously reported the generation of NM ICL mimics by incorporation of ICL precursors into DNA using solid-phase synthesis at defined positions, followed by a double reductive amination reaction. However, the structure of these mimics deviated from the native NM ICLs. Using further development of our approach, we report a new class of NM ICL mimics that only differ from their native counterpart by substitution of dG with 7-deaza-dG at the ICL. Importantly, this approach allows for the synthesis of diverse NM ICLs, illustrated here with a mimic of the adduct formed by chlorambucil. We used the newly generated ICLs in reactions with replicative and translesion synthesis DNA polymerase to demonstrate their stability and utility for functional studies. These new NM ICLs will allow for the further characterization of the biological responses to this important class of antitumor agents.

Bypass of DNA interstrand crosslinks by a Rev1-DNA polymerase ζ complex.

DNA polymerase ζ (Pol ζ) and Rev1 are essential for the repair of DNA interstrand crosslink (ICL) damage. We have used yeast DNA polymerases η, ζ and Rev1 to study translesion synthesis (TLS) past a nitrogen mustard-based interstrand crosslink (ICL) with an 8-atom linker between the crosslinked bases. The Rev1-Pol ζ complex was most efficient in complete bypass synthesis, by 2-3 fold, compared to Pol ζ alone or Pol η. Rev1 protein, but not its catalytic activity, was required for efficient TLS. A dCMP residue was faithfully inserted across the ICL-G by Pol η, Pol ζ, and Rev1-Pol ζ. Rev1-Pol ζ, and particularly Pol ζ alone showed a tendency to stall before the ICL, whereas Pol η stalled just after insertion across the ICL. The stalling of Pol η directly past the ICL is attributed to its autoinhibitory activity, caused by elongation of the short ICL-unhooked oligonucleotide (a six-mer in our study) by Pol η providing a barrier to further elongation of the correct primer. No stalling by Rev1-Pol ζ directly past the ICL was observed, suggesting that the proposed function of Pol ζ as an extender DNA polymerase is also required for ICL repair.