Interstrand crosslink

Interstrand Crosslink Repair
DNA interstrand crosslinks (ICLs) are lesions that covalently link two DNA strands and are extraordinarily cytotoxic since they block essential aspects of DNA metabolism such as replication and transcription. This property is exploited in cancer chemotherapy and ICL-inducing agents such as cisplatin and nitrogen mustards are among the most frequently used antitumor agents used in the clinic. ICLs are also formed by endogenous or exogenous damaging agents such as lipid peroxidation products or formaldehyde. The cellular responses to ICLs, in particular DNA repair, are therefore of importance for maintaining genome stability, but can also lead to resistance of tumor cells against chemotherapeutic agents and are therefore targets for drug design. We are interested in exploring the understanding of ICL repair in healthy cells as well as how ICL repair might be targeted for antitumor drug design.

Synthesis and structural characterization of defined, site-specific ICLs The synthesis of defined ICLs has been of the main hurdles in exploring the cellular responses induced by theses agents. We have developed new synthetic methodology to overcome this limitation. Our strategy is based on the incorporation of appropriate ICL precursors on opposite strands of DNA and the use of a specific coupling reaction to establish the ICL. Using this method we have synthesized ICLs mimicking those formed by nitrogen mustards and chloroethylnitrosoureas. In the nitrogen mustard series our methodology allows for the synthesis of structurally diverse ICL that induce different amount of strain and bending in the ICLs. We are currently studying the structure and dynamic properties of these various ICLs using molecular dynamics simulations and NMR. The availability of structurally diverse ICLs will make it possible to systematically investigate how the structure of ICLs influences the biological responses they trigger.


Uncrosslinked DNA	Nitrogen-mustard mimick	Nitrogen-mustard

Replication dependent and independent ICL repair pathways Using these synthetic adducts we are investigating how these ICLs are processed in mammalian cells using biochemical and cell biological approaches. The repair of ICL is not well understood in eukaryotes, but involves a replication- and recombination-dependent pathway in the S/G2 phases of the cell cycle and a recombination-independent way in G1. We have employed a reporter assay to study recombination-independent ICL repair and found that this repair pathway is dependent on transcription, NER and translesion synthesis. We are presently investigating the transcription-coupled responses to ICL and the synthesis past ICLs by various translesion synthesis polymerases at the biochemical level. The detailed understanding of ICL repair should lead to targets for drug design that might be used to improve existing treatment modalities used in cancer chemotherapy.