Abstract
Elucidation of the function of synaptonemal complex (SC) in Saccharomyces cerevisiae has mainly focused on in vivo analysis of recombination-defective meiotic mutants. Consequently, significant gaps remain in the mechanistic understanding of the activities of various SC proteins and the functional relationships among them. S. cerevisiae Hop1 and Red1 are essential structural components of the SC axial/lateral elements. Previous studies have demonstrated that Hop1 is a structure-selective DNA-binding protein exhibiting high affinity for the Holliday junction and promoting DNA bridging, condensation, and pairing between double-stranded DNA molecules. However, the exact mode of action of Red1 remains unclear, although it is known to interact with Hop1 and to suppress the spore viability defects of hop1 mutant alleles. Here, we report the purification and functional characterization of the full-length Red1 protein. Our results revealed that Red1 forms a stable complex with Hop1 in vitro and provided quantitative insights into their physical interactions. Mechanistically, Red1 preferentially associated with the Holliday junction and 3-way junction rather than with single- or double-stranded DNA with overhangs. Although Hop1 and Red1 exhibited similar binding affinities toward several DNA substrates, the two proteins displayed some significant differences. Notably, Red1, by itself, lacked DNA-pairing ability; however, it potentiated Hop1-promoted intermolecular pairing between double-stranded DNA molecules. Moreover, Red1 exhibited nonhomologous DNA end-joining activity, thus revealing an unexpected role for Red1 in recombination-based DNA repair. Collectively, this study presents the first direct insights into Red1's mode of action and into the mechanism underlying its role in chromosome synapsis and recombination.
Highlights
Elucidation of the function of synaptonemal complex (SC) in Saccharomyces cerevisiae has mainly focused on in vivo analysis of recombination-defective meiotic mutants
The band corresponding to 95.5 kDa was undetectable in the whole-cell lysates of cells grown in the absence of isopropyl -D-1-thiogalactopyranoside
This premise was corroborated by Western blot analysis using anti-Red1 antibodies (Fig. 1B)
Summary
Elucidation of the function of synaptonemal complex (SC) in Saccharomyces cerevisiae has mainly focused on in vivo analysis of recombination-defective meiotic mutants. Hop and Red interact with each other, physically and functionally [12, 39, 43, 46], and bind to SUMO chains prior to the initiation of meiotic interhomolog recombination and chromosome synapsis [47, 48] These studies suggest the importance of Red in meiosis, very little is known about the biochemical properties of Red. In contrast to Hop, Red, by itself, lacks the ability to promote intermolecular pairing between duplex DNA molecules but potentiates the pairing promoted by Hop1 These results are consistent with the idea that Red binding protects DNA structures that are obligatory meiotic recombination intermediates, and its DNA end–joining activity contributes to the maintenance of genome integrity during meiotic chromosome synapsis and recombination
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