Yeast-based assays for the functional characterization of cancer-associated variants of human DNA repair genes.

Tiziana Cervelli,Alvaro Galli,Francesca Bellè,Samuele Lodovichi

doi:10.15698/mic2020.07.721

Abstract

Technological advances are continuously revealing new genetic variants that are often difficult to interpret. As one of the most genetically tractable model organisms, yeast can have a central role in determining the consequences of human genetic variation. DNA repair gene mutations are associated with many types of cancers, therefore the evaluation of the functional impact of these mutations is crucial for risk assessment and for determining therapeutic strategies. Owing to the evolutionary conservation of DNA repair pathways between human cells and the yeast Saccharomyces cerevisiae, several functional assays have been developed. Here, we describe assays for variants of human genes belonging to the major DNA repair pathways divided in functional assays for human genes with yeast orthologues and human genes lacking a yeast orthologue. Human genes with orthologues can be studied by introducing the correspondent human mutations directly in the yeast gene or expressing the human gene carrying the mutations; while the only possible approach for human genes without a yeast orthologue is the heterologous expression. The common principle of these approaches is that the mutated gene determines a phenotypic alteration that can vary according to the gene studied and the domain of the protein. Here, we show how the versatility of yeast can help in classifying cancer-associated variants.

Highlights

hereditary nonpolyposis colorectal cancer (HNPCC) is caused by inactivating mutations in Mismatch Repair (MMR) genes such as MSH2, MSH6, MLH1, PMS1 and PMS2 while breast and ovarian cancer syndrome (HBOC) is caused by inactivating mutations in genes involved in Homologous Recombination (HR) such as BRCA1, BRCA2 [6, 7]
Cancer-associated variants of BRCA1 and the MMR genes MSH2, MLH1 are the most studied in yeast and several functional assays have been developed in order to classify variant of unknown significance” (VUS)
Functional assays to assess the impact of mutations of MMR genes are based on the evaluation of the effect of mutations on the mutator phenotype i.e. the pathogenic variants should affect mutation rate as compared to the wild type (WT)

Summary

Introduction

To determine the functional impact of human DNA repair gene variants, currently available yeast assays are based on the evaluation of forward and reverse mutation, DNA damage sensitivity, transcriptional activity, growth defect, protein mislocalization, intra- and inter-chromosomal recombination (Table1). As the Rad51 and Rad52 protein sequences are highly conserved in human and S. cerevisiae, the functional impact of rare cancer-associated missense variants in both HR repair genes was evaluated by constructing yeast strains carrying correspondent mutated alleles (Figure 1A)[49].

Results

Conclusion