RESUMO
During mismatch repair (MMR) MSH proteins bind to mismatches that form as the result of DNA replication errors and recruit MLH factors such as Mlh1-Pms1 to initiate excision and repair steps. Previously, we identified a negative epistatic interaction involving naturally occurring polymorphisms in the MLH1 and PMS1 genes of baker's yeast. Here we hypothesize that a mutagenic state resulting from this negative epistatic interaction increases the likelihood of obtaining beneficial mutations that can promote adaptation to stress conditions. We tested this by stressing yeast strains bearing mutagenic (incompatible) and non-mutagenic (compatible) mismatch repair genotypes. Our data show that incompatible populations adapted more rapidly and without an apparent fitness cost to high salt stress. The fitness advantage of incompatible populations was rapid but disappeared over time. The fitness gains in both compatible and incompatible strains were due primarily to mutations in PMR1 that appeared earlier in incompatible evolving populations. These data demonstrate a rapid and reversible role (by mating) for genetic incompatibilities in accelerating adaptation in eukaryotes. They also provide an approach to link experimental studies to observational population genomics.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Transporte/genética , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Tolerância ao Sal/genética , ATPases Transportadoras de Cálcio/genética , Replicação do DNA/genética , Chaperonas Moleculares , Proteína 1 Homóloga a MutL , Proteínas MutL , Pressão Osmótica/fisiologia , Saccharomyces cerevisiae/genética , Cloreto de Sódio/metabolismoRESUMO
In budding yeast the pachytene checkpoint 2 (Pch2) protein regulates meiotic chromosome axis structure by maintaining the domain-like organization of the synaptonemal complex proteins homolog pairing 1 (Hop1) and molecular zipper 1 (Zip1). Pch2 has also been shown to modulate meiotic double-strand break repair outcomes to favor recombination between homologs, play an important role in the progression of meiotic recombination, and maintain ribosomal DNA stability. Pch2 homologs are present in fruit flies, worms, and mammals, however the molecular mechanism of Pch2 function is unknown. In this study we provide a unique and detailed biochemical analysis of Pch2. We find that purified Pch2 is an AAA+ (ATPases associated with diverse cellular activities) protein that oligomerizes into single hexameric rings in the presence of nucleotides. In addition, we show Pch2 binds to Hop1, a critical axial component of the synaptonemal complex that establishes interhomolog repair bias, in a nucleotide-dependent fashion. Importantly, we demonstrate that Pch2 displaces Hop1 from large DNA substrates and that both ATP binding and hydrolysis by Pch2 are required for Pch2-Hop1 transactions. Based on these and previous cell biological observations, we suggest that Pch2 impacts meiotic chromosome function by directly regulating Hop1 localization.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Algoritmos , Sequência de Aminoácidos , Cromossomos/ultraestrutura , DNA/química , Teste de Complementação Genética , Glutationa Transferase/metabolismo , Hidrólise , Meiose , Dados de Sequência Molecular , Mutação , Ligação Proteica , Saccharomyces cerevisiae/metabolismoRESUMO
The ability of proteins to locate specific targets among a vast excess of nonspecific DNA is a fundamental theme in biology. Basic principles governing these search mechanisms remain poorly understood, and no study has provided direct visualization of single proteins searching for and engaging target sites. Here we use the postreplicative mismatch repair proteins MutSα and MutLα as model systems for understanding diffusion-based target searches. Using single-molecule microscopy, we directly visualize MutSα as it searches for DNA lesions, MutLα as it searches for lesion-bound MutSα, and the MutSα/MutLα complex as it scans the flanking DNA. We also show that MutLα undergoes intersite transfer between juxtaposed DNA segments while searching for lesion-bound MutSα, but this activity is suppressed upon association with MutSα, ensuring that MutS/MutL remains associated with the damage-bearing strand while scanning the flanking DNA. Our findings highlight a hierarchy of lesion- and ATP-dependent transitions involving both MutSα and MutLα, and help establish how different modes of diffusion can be used during recognition and repair of damaged DNA.