DNA interstrand crosslinks (ICLs) are induced by a number of bifunctional antitumor drugs such as cisplatin, mitomycin C, or the nitrogen mustards as well as endogenous agents formed by lipid peroxidation. The repair of ICLs requires the coordinated interplay of a number of genome maintenance pathways, leading to the removal of ICLs through at least two distinct mechanisms. The major pathway of ICL repair is dependent on replication, homologous recombination, and the Fanconi anemia (FA) pathway, whereas a minor, G0/G1-specific and recombination-independent pathway depends on nucleotide excision repair. A central step in both pathways in vertebrates is translesion synthesis (TLS) and mutants in the TLS polymerases Rev1 and Pol zeta are exquisitely sensitive to crosslinking agents. Here, we review the involvement of Rev1 and Pol zeta as well as additional TLS polymerases, in particular, Pol eta, Pol kappa, Pol iota, and Pol nu, in ICL repair. Biochemical studies suggest that multiple TLS polymerases have the ability to bypass ICLs and that the extent ofbypass depends upon the structure as well as the extent of endo- or exonucleolytic processing of the ICL. As has been observed for lesions that affect only one strand of DNA, TLS polymerases are recruited by ubiquitinated proliferating nuclear antigen (PCNA) to repair ICLs in the G0/G1 pathway. By contrast, this data suggest that a different mechanism involving the FA pathway is operative in coordinating TLS in the context of replication-dependent ICL repair.

DNA interstrand crosslinks (ICLs), inhibit DNA metabolism by covalently linking two strands of DNA and are formed by antitumor agents such as cisplatin and nitrogen mustards. Multiple complex repair pathways of ICLs exist in humans that share translesion synthesis (TLS) past a partially processed ICL as a common step. We have generated site-specific major groove ICLs and studied the ability of Y-family polymerases and Pol ζ to bypass ICLs that induce different degrees of distortion in DNA. Two main factors influenced the efficiency of ICL bypass: the length of the dsDNA flanking the ICL and the length of the crosslink bridging two bases. Our study shows that ICLs can readily be bypassed by TLS polymerases if they are appropriately processed and that the structure of the ICL influences which polymerases are able to read through it.

Plasmids containing a site-specific DNA interstrand cross-link (ICL) are invaluable tools for the investigation of ICL repair pathways at the biochemical and cellular level. We describe a procedure for preparation of plasmid DNA substrates containing a single ICL at a specific site. The procedure is versatile, leads to reliable yields of pure DNA substrate, and is suitable for the incorporation of virtually any type of DNA lesion into plasmids.