Saccharomyces cerevisiae Red1 protein exhibits nonhomologous DNA end joining activity and potentiates Hop1 promoted pairing of double-stranded DNA

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 different 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-specific DNA-binding protein that exhibits high affinity for the Holliday junction, promotes DNA bridging, condensation and pairing between duplex DNA molecules. However, the exact mode of action of Red1 remains unknown, although it interacts with Hop1 and suppresses spore inviability defects of <hop1> mutant allele. Here, we report the first purification and functional characterization of full-length Red1 protein. The results reveal that Red1 forms a stable complex with Hop1 in vitro; the study provides quantitative insights into their physical interactions. Mechanistically, Red1 preferentially associates with the Holliday junction and 3-way junction compared to the single- or duplex DNA with overhangs. Although Hop1 and Red1 exhibit similar binding affinities towards several DNA substrates, significant differences were found between the two proteins. Notably, Red1, by itself, lacks DNA pairing ability; however, it potentiates Hop1 promoted intermolecular pairing between duplex DNA molecules. Moreover, Red1 exhibits non-homologous DNA end-joining activity, thus revealing an unexpected role for Red1 in recombinational DNA repair. Altogether, this study presents the first direct evidence into the mode of action of Red1 and insights into the mechanism underlying its role in chromosome synapsis and recombination.