Genetic interaction analysis of point mutations enables interrogation of gene function at a residue‐level resolution

Abstract

We have achieved a residue-level resolution of genetic interaction mapping – a technique that measures how the function of one gene is affected by the alteration of a second gene – by analyzing point mutations. Here, we describe how to interpret point mutant genetic interactions, and outline key applications for the approach, including interrogation of protein interaction interfaces and active sites, and examination of post-translational modifications. Genetic interaction analysis has proven effective for characterizing cellular processes; however, to date, systematic high-throughput genetic interaction screens have relied on gene deletions or knockdowns, which limits the resolution of gene function analysis and poses problems for multifunctional genes. Our point mutant approach addresses these issues, and further provides a tool for in vivo structure-function analysis that complements traditional biophysical methods. We also discuss the potential for genetic interaction mapping of point mutations in human cells and its application to personalized medicine.

From systems to structure — using genetic data to model protein structures

“…The first pE-MAP covered 53 budding yeast point mutants in RNA polymerase II (RNAPII), crossed against a library of 1,200 deletion and knockdown mutants 89 . This study revealed that pairs of residues that exhibited similar genetic interaction profiles were typically close in space, whether they resided in the same or different RNAPII subunits 89 , 90 . Several early DMS studies revealed similar patterns for the pairwise genetic interactions between point mutants 92 – 94 .…”

“…These results indicated that genetic interactions provide a high level of resolution and allow the dissection of multifunctional proteins into regions that are functionally and physically connected to other factors. Spurred by these findings, the E-MAP technology was extended to screen entire libraries of point mutations in a set of related proteins to generate point mutant E-MAPs (pE-MAPs) 89 , 90 . Quantitative SGA screens have also included large numbers of point mutations; however, these have generally been chosen on the basis of their phenotype as temperature-sensitive alleles of essential genes, rather than systematic mutations of a specific protein or complex 68 , 69 .…”

The Role of S. cerevisiae Sub1/PC4 in Transcription Elongation Depends on the C-Terminal Region and Is Independent of the ssDNA Binding Domain

“…Remarkably, though Sub1 is an elongation factor, the SUB1 deletion mutant is resistant to 6AU because the IMD2 gene is constitutively expressed due to defects in the transcription start site selection. On the contrary, in isogenic wt cells, IMD2 is induced only upon 6AU treatment [ 22 , 26 , 52 ]. Interestingly, we showed that Sub1 influences IMD2 transcription elongation, thereby affecting the novo synthesis of IMD2 after 6AU treatment [ 26 ].…”

Predicting chemotherapeutic drug combinations through gene network profiling

“…With the completion of sequencing for many organisms, especially that of yeast, the ability to interrogate the functional implications of individual genes to a phenotype has revolutionized genome-based studies 25 51 . Large-scale genetic interaction assays have made it possible to model the lives of eukaryotic organisms in order to identify the complicated connectivity among genes and study how this interactivity constitutes a network that determines the phenotype 52 53 ; in this case, chemotherapeutic resistance. The study of genetic interactions has gained momentum, and the revelation of the ‘interactome’ 54 has shed light on how multiple components can be regulated concurrently.…”