Abstract
The evolutionarily conserved Swi5-Sfr1 complex plays an important role in homologous recombination, a process crucial for the maintenance of genomic integrity. Here, we purified Schizosaccharomyces pombe Swi5-Sfr1 complex from meiotic cells and analyzed it by mass spectrometry. Our analysis revealed new phosphorylation sites on Swi5 and Sfr1. We found that mutations that prevent phosphorylation of Swi5 and Sfr1 do not impair their function but swi5 and sfr1 mutants encoding phosphomimetic aspartate at the identified phosphorylation sites are only partially functional. We conclude that during meiosis, Swi5 associates with Sfr1 and both Swi5 and Sfr1 proteins are phosphorylated. However, the functional relevance of Swi5 and Sfr1 phosphorylation remains to be determined.
Highlights
Homologous recombination is a conserved process for repairing several types of lesions, including DNA double-strand breaks [1]
We used the tandem affinity purification (TAP) protocol to purify Swi5-TAP and Sfr1-TAP from cycling S. pombe cells and found that Swi5 was phosphorylated on serine 72 and Sfr1 contained three phosphorylated serine residues (S26, S109 and S165) [24]
To identify additional phosphorylation sites, we mutated the five serine residues found to be phosphorylated in our Sfr1-TAP purifications (S26, S33, S109, S155 and S165) to alanine, which can no longer be phosphorylated and purified Sfr1-5A-TAP from haploid meiotic cells
Summary
Homologous recombination is a conserved process for repairing several types of lesions, including DNA double-strand breaks [1]. Swi5-Sfr1C forms a kinked structure that is able to stimulate Rad51-mediated strand exchange. Swi and Sfr play an important role during meiosis, a specialized cell division that generates gametes with a haploid set of chromosomes from a diploid precursor [9]. This reduction in chromosome number results from one round of DNA replication followed by two nuclear divisions, meiosis I and meiosis II. We found that the identified phosphorylation sites are not essential for the function of Swi and Sfr, phosphomimetic swi and sfr mutants are only partially functional
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