Replication Bypass of DNAs containing Interstrand DNA Crosslinks
One of the current models for repair of DNA interstrand crosslinks (ICLs) proposes a role for translesion synthesis (TLS) DNA polymerases to fill a gap created by dual incision in one of the two affected strands. To gain support for this model, we have constructed DNAs containing site-specific ICLs in which the linkage was between N2 positions of guanines, similar to ICLs formed by mitomycin C (MMC) and bifunctional enals. After engineering model acrolein-mediated N2-N2-guanine ICLs into plasmids and replicating them through mammalian cells, analyses of the mutation spectra revealed that they were only marginally miscoding. To identify the DNA polymerase(s) that could be involved in accurate TLS, a series of oligodeoxy-nucleotides were constructed mimicking potential intermediates in ICL repair. Analyses revealed that although Rev1 could incorporate a dCTP opposite the crosslinked guanine, no evidence was found for TLS by pol ζ or pol ζ /Rev1 combination. In contrast, human pol κ not only catalyzed high fidelity incorporation opposite the crosslinked guanine, but also replicated beyond the lesion. The efficiency of TLS was greatly enhanced by truncation of both the 5´ and 3´ ends of the preincised strand. Biological support for pol κ in TLS past N2-N2-guanine ICLs was demonstrated by both cell survival and chromosomal stability being adversely affected in pol κ-depleted cells following MMC exposure. These studies have now been extended to analyses of the E. coli polymerases in which DNA polymerase IV is able to readily bypass these ICLs with very high fidelity and efficiency. In vivo data demonstrate an absolute dependence on polymerase IV in replicating plasmids containing N2-N2-guanine ICLs.
The data described above suggest that pol κ is essential for restoring crosslink-containing DNAs to a condition in which normal replication, transcription and homologous recombination can occur. Such an activity would limit the effectiveness of crosslink-inducing chemotherapeutic agents to kill targeted cancer cells. Therefore, there is an urgent need for development of new therapies that do not allow cancer cells to repair crosslinks and avoid being killed by these agents. We have made it a priority to identify small molecule inhibitors targeting pol k as crucial for improving the therapeutic efficacy of chemotherapeutic agents.
Specifically, it is hypothesized that pol k specific inhibitors, given in conjunction with crosslinking agents, will increase the therapeutic effectiveness of crosslink-inducing drugs. To identify pol k inhibitors, preliminary high throughput screens have been conducted on 16,000 compounds in collaboration with the NIH Chemical Genomics Center (NCGC) using a fluorescence-based assay. Preliminary candidates were identified and verified using a secondary assay that confirmed the robust nature of high throughput screens to identify potential therapeutic agents. The future aims of this investigation will screen the ~400,000-member Molecular Libraries Small Molecule Repository (MLSMR) collection and analyze inhibitor effectiveness in biological assays.
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