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

Area of Science:

• Genetics
• Molecular Biology
• Genomic Instability

Background:

• Replication Factor A (RFA) is crucial for DNA replication and repair.
• RFA dosage insufficiency can lead to genomic instability.
• Understanding RFA's role is key to comprehending DNA replication stress responses.

Purpose of the Study:

• To investigate genomic alterations in Saccharomyces cerevisiae due to RFA dosage insufficiency.
• To elucidate the mechanisms by which RFA deficiency impacts DNA integrity.
• To explore adaptive strategies cells employ to survive RFA-induced replication stress.

Main Methods:

• Promoter-replacement strategy to control RFA gene expression.
• Mutation accumulation experiments.
• Whole-genome sequencing to identify mutations.
• APOBEC3B-induced mutagenesis assay.
• Analysis of DNA repair gene inactivation.

Main Results:

• Transcriptional suppression of RFA2 or RFA3 causes severe growth defects.
• RFA deficiency leads to increased monosomy, terminal deletions, and loss of heterozygosity.
• Accumulation of exposed single-stranded DNA (ssDNA) biased towards the lagging strand.
• Spontaneous inactivation of Mismatch Repair (MMR) genes (MSH2, PMS1) confers a survival advantage.
• Hypermutation phenotype allows for partial growth recovery.

Conclusions:

• RFA dosage is critical for maintaining genomic integrity.
• Replication stress induces specific mutational signatures.
• Cells modulate DNA repair pathways, like MMR, as an adaptive response to RFA insufficiency.
• This study reveals a link between RFA dosage, replication stress, and adaptive evolution.