Using synthetic DNA interstrand crosslinks to elucidate repair pathways and identify new therapeutic targets for cancer chemotherapy

Abstract

Many cancer chemotherapeutic agents form DNA interstrand crosslinks (ICLs), extremely cytotoxic lesions that form covalent bonds between two opposing DNA strands, blocking DNA replication and transcription. However, cellular responses triggered by ICLs can cause resistance in tumor cells, limiting the efficacy of such treatment. Here we discuss recent advances in our understanding of the mechanisms of ICL repair that cause this resistance. The recent development of strategies for the synthesis of site-specific ICLs greatly contributed to these insights. Key features of repair are similar for all ICLs, but there is increasing evidence that the specifics of lesion recognition and synthesis past ICLs by DNA polymerases are dependent upon the structure of ICLs. These new insights provide a basis for the improvement of antitumor therapy by targeting DNA repair pathways that lead to resistance to treatment with crosslinking agents.

Fig. 1

Methods for the preparation of DNA interstrand crosslinks. a Treatment of an oligonucleotide with a bifunctional alkylating agent. This method gives rise to a mixture of products (intra- and interstrand crosslinks, monoadducts) and ICL typically make up less than 5% of all the products. b Two-step ICL formation. After reaction of a single strand containing one guanine residue, the monoadduct is purified, annealed with a complementary strand, and activated to react with the other strand. This method is more efficient than a, but yields typically do not exceed 20%. c Multi-step solid-phase synthesis; the crosslink is chemically synthesized as a nucleotide dimer and incorporated into DNA followed by bidirectional solid-phase DNA synthesis. This approach yields highly specific ICLs. d Two crosslink precursors are incorporated into DNA using solid-phase DNA synthesis and the ICL formed by a selective post-synthetic crosslinking reaction. This approach also yields ICLs with high specificity and in high yields. ICLs are shown as red diamonds

Fig. 2

Common ICL-forming agents and their DNA adducts. a Although native BCNU adducts have not yet been synthesized, various mimics have been synthesized and used to study repair. The structure of the ICL and the pdb file refer to one of these mimics. b This ICL has been synthesized in two sequence contexts. This ICL is a reduced and stabilized form of the crosslinks formed by malondialdehyde. c The structure shown here is based on a molecular modeling study [28]. d The coordinates for the mitomycin C ICL were kindly provided by Suse Broyde based on Ref [51]. e The distortion refers to the mimic showed here. The exact distortion provoked by BCNU is not known and is likely to be different than the mimic

Fig. 3

Models for replication-dependent (left) and -independent (right) ICL repair pathways. In the replication-dependent pathway, the lesion is detected when replication forks are blocked by the ICL. The replication fork initially pauses 20–40 nucleotides from the ICLs, and then approaches to the ICL, activating the FA pathway. The FA core complex ubiquitinates the FANCD2–FANCI complex, a step required for the endonucleases to incise the lagging strand on both sides of the ICL and REV1 and Polζ to extend the leading strand past the ICL. It is believed that HR and possibly NER then complete the process and restore the intact DNA sequence. In G0/G1, the ICL can also be recognized by an RNA polymerase during transcription or, depending on its structure by the NER damage recognition factor XPC-RAD23B. NER proteins are thought to be responsible to unhook the ICL initiating repair synthesis and carrying out TLS past the unhooked ICL in a manner dependent on ubiquitinated PCNA and a TLS polymerase (probably Rev1 and Polζ to bypass the lesion in an potentially error-prone manner. Finally, it is believed that the NER machinery eliminates the ICL remnant restoring the intact DNA