Genomic Instability and Adaptive Evolution Induced by RFA Insufficiency in Saccharomyces cerevisiae

TL;DR

This study shows how reduced RFA levels in yeast cause genomic instability and adaptive evolution through specific mutations and repair pathway changes.

Contribution

The study reveals RFA insufficiency's role in inducing a unique mutational spectrum and adaptive MMR gene inactivation in yeast.

Findings

RFA deficiency causes monosomy and terminal deletions, indicating replication stress.

Loss of heterozygosity is enriched at centromeres and high-GC regions.

MMR gene inactivation helps yeast survive RFA stress and recover growth.

Abstract

This study systematically investigated the genomic alterations in Saccharomyces cerevisiae driven by Replication Factor A (RFA) dosage insufficiency using a promoter-replacement strategy combined with mutation accumulation and whole-genome sequencing. Our findings reveal that transcriptional suppression of RFA2 or RFA3 leads to severe growth inhibition. RFA deficiency induces a distinct mutational spectrum characterized by a high frequency of monosomy and terminal deletions, indicative of severe replication stress. Furthermore, loss of heterozygosity is significantly enriched at centromeres and high-GC regions, underscoring the role of RFA in stabilizing intrinsic genomic barriers. Utilizing an APOBEC3B-induced mutagenesis assay, we demonstrate that RFA insufficiency leads to the extensive accumulation of exposed ssDNA with a distinct bias towards the lagging strand template. Notably, we observed that cells spontaneously inactivate Mismatch Repair (MMR) genes, such as MSH2 and PMS1, to survive RFA-induced stress. This hypermutant phenotype grants a certain degree of growth recovery on Low Galactose (LG) medium. Overall, these findings demonstrate that RFA dosage is a key determinant of genomic integrity and elucidate how repair pathway modulation drives adaptive evolution under replication stress.

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