Eukaryotic Rad51 protein is essential for homologous-recombination repair of DNA double-strand breaks. Rad51 recombinases first assemble onto single-stranded DNA to form a nucleoprotein filament, required for function in homology pairing and strand exchange. This filament assembly is the first regulation step in homologous recombination. Rad51 nucleation is kinetically slow, and several accessory factors have been identified to regulate this step. Swi5–Sfr1 (S5S1) stimulates Rad51-mediated homologous recombination by stabilizing Rad51 nucleoprotein filaments, but the mechanism of stabilization is unclear. We used single-molecule tethered particle motion experiments to show that mouse S5S1 (mS5S1) efficiently stimulates mouse RAD51 (mRAD51) nucleus formation and inhibits mRAD51 dissociation from filaments. We also used single-molecule fluorescence resonance energy transfer experiments to show that mS5S1 promotes stable nucleus formation by specifically preventing mRAD51 dissociation. This leads to a reduction of nucleation size from three mRAD51 to two mRAD51 molecules in the presence of mS5S1. Compared with mRAD51, fission yeast Rad51 (SpRad51) exhibits fast nucleation but quickly dissociates from the filament. SpS5S1 specifically reduces SpRad51 disassembly to maintain a stable filament. These results clearly demonstrate the conserved function of S5S1 by primarily stabilizing Rad51 on DNA, allowing both the formation of the stable nucleus and the maintenance of filament length.

E. coli RecA recombinase catalyzes the homology pairing and strand exchange reactions in homologous recombinational repair. RecA must compete with single-stranded DNA binding proteins (SSB) for single-stranded DNA (ssDNA) substrates to form RecA nucleoprotein filaments, as the first step of this repair process. It has been suggested that RecA filaments assemble mainly by binding and extending onto the free ssDNA region not covered by SSB, or are assisted by mediators. Using the tethered particle motion (TPM) technique, we monitored individual RecA filament assembly on SSB-wrapped ssDNA in real-time. Nucleation times of the RecA E38K nucleoprotein filament assembly showed no apparent dependence among DNA substrates with various ssDNA gap lengths (from 60 to 100 nucleotides) wrapped by one SSB in the (SSB)65 binding mode. Our data have shown an unexpected RecA filament assembly mechanism in which a RecA-SSB-ssDNA interaction exists. Four additional pieces of evidence support our claim: the nucleation times of the RecA assembly varied (1) when DNA substrates contained different numbers of bound SSB tetramers; (2) when the SSB wrapping mode conversion is induced; (3) when SSB C-terminus truncation mutants are used; and (4) when an excess of C-terminal peptide of SSB is present. Thus, a RecA-SSB interaction should be included in discussing RecA regulatory mechanism.

The glycocalyx is a shell of heavily glycosylated proteins and lipids distributed on the cell surface of nearly all cell types. Recently, it has been found that bulky transmembrane glycoproteins such as MUC1 can modulate membrane shape by inducing membrane protrusions. In this work, we examine the reciprocal relationship of how membrane shape affects MUC1’s spatial distribution on the cell membrane and its biological significance. By employing nanopatterned surfaces and membrane-sculpting proteins to manipulate membrane curvature, we show that MUC1 avoids positively-curved membranes (membrane invaginations) and accumulates on negatively-curved membranes (membrane protrusions). MUC1’s curvature sensitivity is dependent on the length and the extent of glycosylation of its ectodomain, with large and highly glycosylated forms preferentially staying out of positive curvature. Interestingly, MUC1’s avoidance of positive membrane curvature enables it to escape from endocytosis and being removed from the cell membrane. These findings also suggest that the truncation of MUC1’s ectodomain, often observed in breast and ovarian cancers, may enhance its endocytosis and potentiate its intracellular accumulation and signaling.

Microcephalin 1 (MCPH1) was identified from genetic mutations in patients with primary autosomal recessive microcephaly. In response to DNA double-strand breaks (DSBs), MCPH1 forms damage-induced foci and recruits BRCA2–RAD51 complex, a key component of the DSB repair machinery for homologous recombination (HR), to damage sites. Accordingly, the efficiency of HR is significantly attenuated upon depletion of MCPH1. The biochemical characteristics of MCPH1 and its functional interaction with the HR machinery had remained unclear due to lack of highly purified MCPH1 recombinant protein for functional study. Here, we established a mammalian expression system to express and purify MCPH1 protein. We show that MCPH1 is a bona fide DNA-binding protein and provide direct biochemical analysis of this MCPH family protein. Furthermore, we reveal that MCPH1 directly interacts with RAD51 at multiple contact points, providing evidence for how MCPH1 physically engages with the HR machinery. Importantly, we demonstrate that MCPH1 enhances the stability of RAD51 on single-strand DNA, a prerequisite step for RAD51-mediated recombination. Single-molecule tethered particle motion analysis showed a ∼2-fold increase in the lifetime of RAD51–ssDNA filaments in the presence of MCPH1. Thus, our study demonstrates direct crosstalk between microcephaly protein MCPH1 and the recombination component RAD51 for DSB repair.

DNA topology is thought to affect DNA enzyme activity. The helical structure of duplex DNA dictates the change of topological states during strand separation when DNA is constrained. During the repair of DNA double-stranded breaks, the RecA nucleoprotein filament invades DNA and carries out consecutive strand exchange reactions coupled with duplex DNA strand separation. It has been suggested that torsional strain could be generated and its accumulation could inhibit strand exchange. We used hairpin and nicked DNA substrates to test how torsional strain alters the RecA-mediated strand exchange efficiency. Single-molecule tethered particle motion (TPM) experiments showed that torsionally constrained hairpin DNA substrates returned nearly no successful strand exchange events catalyzed by RecA. Surprisingly, the strand exchange efficiencies increase in the presence of DNA nicks or loop disruption. The dwell time of transient RecA events in hairpin is shorter compared to those found in nicked or fork DNA substrates, which suggests a limited strand exchange progression in hairpin substrates. Our observation shows that RecA generates local torsional strain during strand exchange, and the inability to dissipate this torsional strain inhibits homologous recombination progression. DNA topological states are thus important regulation measures of DNA recombination.