A New Approach in Identifying dss1 Suppressors by Whole Genome Sequencing of Ustilago maydis

Abstract

Dss1, a small and intrinsically disordered protein, is one of the crucial regulators of homologous recombination (HR), a key DNA repair pathway that ensures error-free repair of double-strand breaks, thereby preserving genome stability. Dss1 is highly conserved in eukaryotes and is present in a model organism Ustilago maydis, known for its extreme resistance to ionizing and UV irradiation. This microorganism is a valuable model for investigating HR, which combines high genetic tractability and a DNA-repair system remarkably similar to humans’, including conserved BRCA2 ortholog Brh2 and its partner Dss1. Loss of dss1 function leads to the extreme sensitivity of the U. maydis mutant to genotoxic agents due to impaired DNA-repair and HR. The main aim of the research is to identify novel cellular factors involved in HR by searching for suppressors of dss1, secondary mutations that rescue genotoxic resistance of dss1 mutant. To uncover such suppressors, several independent Δdss1 suppressor strains were generated by random mutagenesis and phenotypically characterized. While complementation cloning, classical genetic approach, could not reveal the suppressor identity, we applied whole-genome sequencing in combination with a modified pooled-linkage analysis. This strategy is based on sequencing DNA from pools of meiotic segregants, grouped according to their expected phenotype. By separating putative suppressor and wt segregants, it becomes possible to track how often specific variant appear in each pool. Variants that consistently co-segregate with the expected suppressor phenotype show a shift in allele frequency, which points to genomic regions that are likely to carry the suppressor mutation. Although the analysis is ongoing, the study demonstrates the applicability of pooled-linkage sequencing as a complementary method to classical genetic tools. The approach has the potential to facilitate the identification of novel factors involved in HR in U. maydis, thus, representing an applicable framework for uncovering genetic networks that maintain genome integrity.Book of abstract: 8th edition of Young Biologists Matter Congress (Molecular Biology & Physiology) Thessaloniki, Greece, 29th September – 4th October 202