dRnh1-GFP and Drug Discovery: A Toolkit for Better Understanding the Mcm2-7 Replicative Helicase

The faithful replication of the DNA code is essential for the propagation of living cells. At the head of this replication process is the DNA helicase Mcm2-7, which serves to unwind DNA’s double-helix structure and provide access for the DNA replication machinery. Though Mcm2-7 has been extensively characterized by biochemical, single-molecule, and structural studies, questions remain regarding its DNA translocation mechanism, as well as in understanding how Mcm2-7 bypasses “road blocks” caused by RNA polymerase. Here, I discuss the development and preliminary implementation of two tools designed in part to better understand these different aspects of Mcm2-7’s function in Saccharomyces cerevisiae. The first such tool is a drug discovery pipeline in which the principle of drug synthetic lethality with an mcm2 mutant (mcm2-DENQ) was used to identify novel Mcm2-7 inhibitors. This high-throughput screen, and its secondary assays, identified N-Methyl- β -carboline-3-carboxamide (CMA) as a likely inhibitor of active Mcm2-7. CMA was found to specifically inhibit growth and cell survival of mcm2-DENQ, arrest S-phase DNA replication, and bind to purified Mcm2-7. With this high-throughput assay and secondary assay pipeline established, more novel inhibitors may be discovered and utilized in mechanistic studies of Mcm2-7 as well as for potential chemotherapeutics. Mcm2-7 mutation can also cause an increase in genomic RNA/DNA hybrids, likely due to an increased rate of unresolved conflicts between transcription and replication machinery. To better understand how Mcm2-7 contributes to/prevents transcription-replication conflicts, I developed a live-cell cytological assay for RNA/DNA hybrid detection in S. cerevisiae. This assay utilizes a catalytically-dead RNaseH1 (dRnh1-GFP) to detect hybrids through formation of easily-detectable nuclear foci. I found that dRnh1-GFP colocalizes with RNA/DNA hybrids as defined by an established immunological assay (S9.6 antibody). Importantly, the percentage of dRnh1-GFP focus-positive cells increased in response to mutations and drug treatments that are known to increase RNA/DNA hybrids, as well as for select drugs known to poison replication forks and promote transcription-replication conflicts. The simplicity and versatility dRnh1-GFP allows for its use outside of Mcm2-7 research and also allows for use in high-throughput screening. With these two tools, we may further our understanding of the Mcm2-7 helicase.