Replication Bypass of DNAs containing Interstrand DNA Crosslinks
Background and Previous Work
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.
Most Recent Work
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 kspecific 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.
Klug AR, Harbut MB, Lloyd RS, Minko IG. Replication Bypass of N(2)-Deoxyguanosine Interstrand Cross-Links by Human DNA Polymerase Eta and Iota. Chem Res Toxicol. 2012 Feb 29 [Epub ahead of print]. PMID: 22332732
Yamanaka K, Minko IG, Takata K, Kolbanovskiy A, Kozekov ID, Wood RD, Rizzo CJ, Lloyd RS. Novel enzymatic function of DNA polymerase nu in translesion DNA synthesis past major groove DNA-peptide and DNA-DNA cross-links. Chem Res Toxicol. 23(3): 689-95, 2010. PMID: 20102227
Kumari, A, Minko, IG, Harbut, MB, Finkel, SE, Goodman, MF, and Lloyd, RS. Replication bypass of interstrand cross-link intermediates by Escherichia coli DNA polymerase IV. J Biol Chem. 283(41):27433-7, 2008. PMID: 18697749
Stone MP, Cho YJ, Huang H, Kim HY, Kozekov ID, Kozekova A, Wang H, Minko IG, Lloyd RS, Harris TM, Rizzo CJ. Interstrand DNA cross-links induced by alpha, beta-unsaturated aldehydes derived from lipid peroxidation and environmental sources. Acc Chem Res. 41(7): 793-804, 2008. PMID: 18500830
Minko IG, Harbut, MB, Kozekov ID, Kozekova A, Jakobs, PM, Olson, SB, Moses, RE, Harris TM, Rizzo CJ, Lloyd RS. Role for DNA polymerase kappa in the processing of N2-N2 guanine interstrand crosslinks. J Biol Chem. 283(25):17075-17082, 2008. PMID: 18434313
Cho YJ, Wang H, Kozekov ID, Kurtz AJ, Jacob J, Voehler M, Smith J, Harris TM, Lloyd RS, Rizzo CJ, Stone MP. Stereospecific formation of interstrand carbinolamine DNA cross-links by crotonaldehyde- and acetaldehyde-derived alpha-CH3-gamma-OH-1,N2-propano-2'-deoxyguanosine adducts in the 5'-CpG-3' sequence. Chem Res Toxicol. 19(2):195-208, 2006. PMID: 16485895
Cho YJ, Kim HY, Huang H, Slutsky A, Minko IG, Wang H, Nechev LV, Kozekov ID, Kozekova A, Tamura P, Jacob J, Voehler M, Harris TM, Lloyd RS, Rizzo CJ, Stone MP. Spectroscopic characterization of interstrand carbinolamine cross-links formed in the 5'-CpG-3' sequence by the acrolein-derived gamma-OH-1,N2-propano-2'-deoxyguanosine DNA adduct. J Am Chem Soc. 127(50):17686-96, 2005. PMID: 16351098
Sanchez AM, Kozekov ID, Harris TM, Lloyd RS. Formation of inter- and intrastrand imine type DNA-DNA cross-links through secondary reactions of aldehydic adducts. Chem Res Toxicol. 18(11):1683-90, 2005. PMID: 16300377