Establishment of DNA-DNA Interactions by the Cohesin Ring.

The ring-shaped structural maintenance of chromosome (SMC) complexes are multi-subunit ATPases that topologically encircle DNA. SMC rings make vital contributions to numerous chromosomal functions, including mitotic chromosome condensation, sister chromatid cohesion, DNA repair, and transcriptional regulation. They are thought to do so by establishing interactions between more than one DNA. Here, we demonstrate DNA-DNA tethering by the purified fission yeast cohesin complex. DNA-bound cohesin efficiently and topologically captures a second DNA, but only if that is single-stranded DNA (ssDNA). Like initial double-stranded DNA (dsDNA) embrace, second ssDNA capture is ATP-dependent, and it strictly requires the cohesin loader complex. Second-ssDNA capture is relatively labile but is converted into stable dsDNA-dsDNA cohesion through DNA synthesis. Our study illustrates second-DNA capture by an SMC complex and provides a molecular model for the establishment of sister chromatid cohesion.

DNA Entry into and Exit out of the Cohesin Ring by an Interlocking Gate Mechanism.

Structural maintenance of chromosome (SMC) complexes are proteinaceous rings that embrace DNA to enable vital chromosomal functions. The ring is formed by two SMC subunits, closed at a pair of ATPase heads, whose interaction is reinforced by a kleisin subunit. Using biochemical analysis of fission-yeast cohesin, we find that a similar series of events facilitates both topological entrapment and release of DNA. DNA-sensing lysines trigger ATP hydrolysis to open the SMC head interface, whereas the Wapl subunit disengages kleisin, but only after ATP rebinds. This suggests an interlocking gate mechanism for DNA transport both into and out of the cohesin ring. The entry direction is facilitated by a cohesin loader that appears to fold cohesin to expose the DNA sensor. Our results provide a model for dynamic DNA binding by all members of the SMC family and explain how lysine acetylation of cohesin establishes enduring sister chromatid cohesion.

Coordination of cohesin and DNA replication observed with purified proteins.

Two newly duplicated copies of genomic DNA are held together by the ring-shaped cohesin complex to ensure faithful inheritance of the genome during cell division1-3. Cohesin mediates sister chromatid cohesion by topologically entrapping two sister DNAs during DNA replication4,5, but how cohesion is established at the replication fork is poorly understood. Here, we studied the interplay between cohesin and replication by reconstituting a functional replisome using purified proteins. Once DNA is encircled before replication, the cohesin ring accommodates replication in its entirety, from initiation to termination, leading to topological capture of newly synthesized DNA. This suggests that topological cohesin loading is a critical molecular prerequisite to cope with replication. Paradoxically, topological loading per se is highly rate limiting and hardly occurs under the replication-competent physiological salt concentration. This inconsistency is resolved by the replisome-associated cohesion establishment factors Chl1 helicase and Ctf4 (refs. 6,7), which promote cohesin loading specifically during continuing replication. Accordingly, we found that bubble DNA, which mimics the state of DNA unwinding, induces topological cohesin loading and this is further promoted by Chl1. Thus, we propose that cohesin converts the initial electrostatic DNA-binding mode to a topological embrace when it encounters unwound DNA structures driven by enzymatic activities including replication. Together, our results show how cohesin initially responds to replication, and provide a molecular model for the establishment of sister chromatid cohesion.

A conserved Ctp1/CtIP C-terminal peptide stimulates Mre11 endonuclease activity.

The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5'-ended DNA strands at DSB ends, producing 3'-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.

Rrp1 translocase and ubiquitin ligase activities restrict the genome destabilising effects of Rad51 in fission yeast.

Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.

Fundamental cell cycle kinases collaborate to ensure timely destruction of the synaptonemal complex during meiosis.

The synaptonemal complex (SC) is a proteinaceous macromolecular assembly that forms during meiotic prophase I and mediates adhesion of paired homologous chromosomes along their entire lengths. Although prompt disassembly of the SC during exit from prophase I is a landmark event of meiosis, the underlying mechanism regulating SC destruction has remained elusive. Here, we show that DDK (Dbf4-dependent Cdc7 kinase) is central to SC destruction. Upon exit from prophase I, Dbf4, the regulatory subunit of DDK, directly associates with and is phosphorylated by the Polo-like kinase Cdc5. In parallel, upregulated CDK1 activity also targets Dbf4. An enhanced Dbf4-Cdc5 interaction pronounced phosphorylation of Dbf4 and accelerated SC destruction, while reduced/abolished Dbf4 phosphorylation hampered destruction of SC proteins. SC destruction relieved meiotic inhibition of the ubiquitous recombinase Rad51, suggesting that the mitotic recombination machinery is reactivated following prophase I exit to repair any persisting meiotic DNA double-strand breaks. Taken together, we propose that the concerted action of DDK, Polo-like kinase, and CDK1 promotes efficient SC destruction at the end of prophase I to ensure faithful inheritance of the genome.

Homology length dictates the requirement for Rad51 and Rad52 in gene targeting in the Basidiomycota yeast Naganishia liquefaciens.

Here, we report the development of methodologies that enable genetic modification of a Basidiomycota yeast, Naganishia liquifaciens. The gene targeting method employs electroporation with PCR products flanked by an 80 bp sequence homologous to the target. The method, combined with a newly devised CRISPR-Cas9 system, routinely achieves 80% gene targeting efficiency. We further explored the genetic requirement for this homologous recombination (HR)-mediated gene targeting. The absence of Ku70, a major component of the non-homologous end joining (NHEJ) pathway of DNA double-strand break repair, almost completely eliminated inaccurate integration of the marker. Gene targeting with short homology (80 bp) was almost exclusively dependent on Rad52, an essential component of HR in the Ascomycota yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. By contrast, the RecA homolog Rad51, which performs homology search and strand exchange in HR, plays a relatively minor role in gene targeting, regardless of the homology length (80 bp or 1 kb). The absence of both Rad51 and Rad52, however, completely eliminated gene targeting. Unlike Ascomycota yeasts, the absence of Rad52 in N. liquefaciens conferred only mild sensitivity to ionizing radiation. These traits associated with the absence of Rad52 are reminiscent of findings in mice.

Dual regulation of Dmc1-driven DNA strand exchange by Swi5-Sfr1 activation and Rad22 inhibition.

Both ubiquitously expressed Rad51 and meiosis-specific Dmc1 are required for crossover production during meiotic recombination. The budding yeast Rad52 and its fission yeast ortholog, Rad22, are "mediators;" i.e., they help load Rad51 onto ssDNA coated with replication protein A (RPA). Here we show that the Swi5-Sfr1 complex from fission yeast is both a mediator that loads Dmc1 onto ssDNA and a direct "activator" of DNA strand exchange by Dmc1. In stark contrast, Rad22 inhibits Dmc1 action by competing for its binding to RPA-coated ssDNA. Thus, Rad22 plays dual roles in regulating meiotic recombination: activating Rad51 and inhibiting Dmc1.

Biochemical reconstitution of topological DNA binding by the cohesin ring.

Cohesion between sister chromatids, mediated by the chromosomal cohesin complex, is a prerequisite for faithful chromosome segregation in mitosis. Cohesin also has vital roles in DNA repair and transcriptional regulation. The ring-shaped cohesin complex is thought to encircle sister DNA strands, but its molecular mechanism of action is poorly understood and the biochemical reconstitution of cohesin activity in vitro has remained an unattained goal. Here we reconstitute cohesin loading onto DNA using purified fission yeast cohesin and its loader complex, Mis4(Scc2)-Ssl3(Scc4) (Schizosaccharomyces pombe gene names appear throughout with their more commonly known Saccharomyces cerevisiae counterparts added in superscript). Incubation of cohesin with DNA leads to spontaneous topological loading, but this remains inefficient. The loader contacts cohesin at multiple sites around the ring circumference, including the hitherto enigmatic Psc3(Scc3) subunit, and stimulates cohesin's ATPase, resulting in efficient topological loading. The in vitro reconstitution of cohesin loading onto DNA provides mechanistic insight into the initial steps of the establishment of sister chromatid cohesion and other chromosomal processes mediated by cohesin.

The fission yeast meiosis-specific Dmc1 recombinase mediates formation and branch migration of Holliday junctions by preferentially promoting strand exchange in a direction opposite to that of Rad51.

Homologous recombination proceeds via the formation of several intermediates including Holliday junctions (HJs), which are important for creating crossover products. DNA strand exchange is a core reaction that produces these intermediates that is directly catalyzed by RecA family recombinases, of which there are two types in eukaryotes: universal Rad51 and meiosis-specific Dmc1. We demonstrated previously that Rad51 promotes four-strand exchange, mimicking the formation and branch migration of HJs. Here we show that Dmc1 from fission yeast has a similar activity, which requires ATP hydrolysis and is independent of an absolute requirement for the Swi5-Sfr1 complex. These features are critically different from three-strand exchange mediated by Dmc1, but similar to those of four-strand exchange mediated by Rad51, suggesting that strand exchange reactions between duplex-duplex and single-duplex DNAs are mechanistically different. Interestingly, despite similarities in protein structure and in reaction features, the preferential polarities of Dmc1 and Rad51 strand exchange are different (Dmc1 promotes exchange in the 5'-to-3' direction and Rad51 promotes exchange in the 3'-to-5' direction relative to the ssDNA region of the DNA substrate). The significance of the Dmc1 polarity is discussed within the context of the necessity for crossover production.

In vitro site-specific recombination mediated by the tyrosine recombinase XerA of Thermoplasma acidophilum.

In organisms with circular chromosomes, such as bacteria and archaea, an odd number of homologous recombination events can generate a chromosome dimer. Such chromosome dimers cannot be segregated unless they are converted to monomers before cell division. In Escherichia coli, dimer-to-monomer conversion is mediated by the paralogous XerC and XerD recombinases at a specific dif site in the replication termination region. Dimer resolution requires the highly conserved cell division protein/chromosome translocase FtsK, and this site-specific chromosome resolution system is present or predicted in most bacteria. However, most archaea have only XerA, a homologue of the bacterial XerC/D proteins, but no homologues of FtsK. In addition, the molecular mechanism of XerA-mediated chromosome resolution in archaea has been less thoroughly elucidated than those of the corresponding bacterial systems. In this study, we identified two XerA-binding sites (dif1 and dif2) in the Thermoplasma acidophilum chromosome. In vitro site-specific recombination assays showed that dif2, but not dif1, serves as a target site for XerA-mediated chromosome resolution. Mutational analysis indicated that not only the core consensus sequence of dif2, but also its flanking regions play important roles in the recognition and recombination reactions mediated by XerA.

Chromosome segregation: how to open cohesin without cutting the ring?

Chromosome segregation is triggered when the ring-shaped chromosomal cohesin complex is opened by proteolytic cleavage to release pairs of sister chromatids. Even before this dramatic event in anaphase, many cohesin rings lead a dynamic life on chromosomes, and in metazoan cells a good part of them dissociate from chromosome arms during mitotic prophase. Two new papers in The EMBO Journal address how chromatin can get into and out of the cohesin ring without requiring its cleavage.

Formation and branch migration of Holliday junctions mediated by eukaryotic recombinases.

Holliday junctions (HJs) are key intermediates in homologous recombination and are especially important for the production of crossover recombinants. Bacterial RecA family proteins promote the formation and branch migration of HJs in vitro by catalysing a reciprocal DNA-strand exchange reaction between two duplex DNA molecules, one of which contains a single-stranded DNA region that is essential for initial nucleoprotein filament formation. This activity has been reported only for prokaryotic RecA family recombinases, although eukaryotic homologues are also essential for HJ production in vivo. Here we show that fission yeast (Rhp51) and human (hRad51) RecA homologues promote duplex-duplex DNA-strand exchange in vitro. As with RecA, a HJ is formed between the two duplex DNA molecules, and reciprocal strand exchange proceeds through branch migration of the HJ. In contrast to RecA, however, strand exchange mediated by eukaryotic recombinases proceeds in the 3'-->5' direction relative to the single-stranded DNA region of the substrate DNA. The opposite polarity of Rhp51 makes it especially suitable for the repair of DNA double-strand breaks, whose repair is initiated at the processed ends of breaks that have protruding 3' termini.

Fission yeast Swi5-Sfr1 protein complex, an activator of Rad51 recombinase, forms an extremely elongated dogleg-shaped structure.

In eukaryotes, DNA strand exchange is the central reaction of homologous recombination, which is promoted by Rad51 recombinases forming a right-handed nucleoprotein filament on single-stranded DNA, also known as a presynaptic filament. Accessory proteins known as recombination mediators are required for the formation of the active presynaptic filament. One such mediator in the fission yeast Schizosaccharomyces pombe is the Swi5-Sfr1 complex, which has been identified as an activator of Rad51 that assists in presynaptic filament formation and stimulates its strand exchange reaction. Here, we determined the 1:1 binding stoichiometry between the two subunits of the Swi5-Sfr1 complex using analytical ultracentrifugation and electrospray ionization mass spectrometry. Small-angle x-ray scattering experiments revealed that the Swi5-Sfr1 complex displays an extremely elongated dogleg-shaped structure in solution, which is consistent with its exceptionally high frictional ratio (f/f(0)) of 2.0 ± 0.2 obtained by analytical ultracentrifugation. Furthermore, we determined a rough topology of the complex by comparing the small-angle x-ray scattering-based structures of the Swi5-Sfr1 complex and four Swi5-Sfr1-Fab complexes, in which the Fab fragments of monoclonal antibodies were specifically bound to experimentally determined sites of Sfr1. We propose a model for how the Swi5-Sfr1 complex binds to the Rad51 filament, in which the Swi5-Sfr1 complex fits into the groove of the Rad51 filament, leading to an active and stable presynaptic filament.

Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators.

In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that two mediators, Rad22 (the S. pombe Rad52 homolog) and the Swi5-Sfr1 complex, participate in a common pathway of Rhp51 (the S. pombe Rad51 homolog)-mediated homologous recombination (HR) and HR repair. Here, we have demonstrated an in vitro reconstitution of the central step of DNA strand exchange during HR. Our system consists entirely of homogeneously purified proteins, including Rhp51, the two mediators, and replication protein A (RPA), which reflects genetic requirements in vivo. Using this system, we present the first robust biochemical evidence that concerted action of the two mediators directs the loading of Rhp51 onto single-stranded DNA (ssDNA) precoated with RPA. Dissection of the reaction reveals that Rad22 overcomes the inhibitory effect of RPA on Rhp51-Swi5-Sfr1-mediated strand exchange. In addition, Rad22 negates the requirement for a strict order of protein addition to the in vitro system. However, despite the presence of Rad22, Swi5-Sfr1 is still essential for strand exchange. Importantly, Rhp51, but neither Rad22 nor the Swi5-Sfr1 mediator, is the factor that displaces RPA from ssDNA. Swi5-Sfr1 stabilizes Rhp51-ssDNA filaments in an ATP-dependent manner, and this stabilization is correlated with activation of Rhp51 for the strand exchange reaction. Rad22 alone cannot activate the Rhp51 presynaptic filament. AMP-PNP, a nonhydrolyzable ATP analog, induces a similar stabilization of Rhp51, but this stabilization is independent of Swi5-Sfr1. However, hydrolysis of ATP is required for processive strand transfer, which results in the formation of a long heteroduplex. Our in vitro reconstitution system has revealed that the two mediators have indispensable, but distinct, roles for mediating Rhp51 loading onto RPA-precoated ssDNA.

The Swi5-Sfr1 complex stimulates Rhp51/Rad51- and Dmc1-mediated DNA strand exchange in vitro.

Nucleoprotein filaments made up of Rad51 or Dmc1 recombinases, the core structures of recombination, engage in ATP-dependent DNA-strand exchange. The ability of recombinases to form filaments is enhanced by recombination factors termed 'mediators'. Here, we show that the Schizosaccharomyces pombe Swi5-Sfr1 complex, a conserved eukaryotic protein complex, at substoichiometric concentrations stimulates strand exchange mediated by Rhp51 (the S. pombe Rad51 homolog) and Dmc1 on long DNA substrates. Reactions mediated by both recombinases are completely dependent on Swi5-Sfr1, replication protein A (RPA) and ATP, although RPA inhibits the reaction when it is incubated with single-stranded DNA (ssDNA) before the recombinase. The Swi5-Sfr1 complex overcomes, at least partly, the inhibitory effect of RPA, representing a novel class of mediator. Notably, the Swi5-Sfr1 complex preferentially stimulates the ssDNA-dependent ATPase activity of Rhp51, and it increases the amounts of Dmc1 bound to ssDNA.

Expression, purification and crystallization of Swi5 and the Swi5-Sfr1 complex from fission yeast.

The assembly of the presynaptic filament of recombinases represents the most important step in homologous recombination. The formation of the filament requires assistance from mediator proteins. Swi5 and Sfr1 have been identified as mediators in fission yeast and these proteins form a complex that stimulates strand exchange. Here, the expression, purification and crystallization of Swi5 and its complex with an N-terminally truncated form of Sfr1 (DeltaN180Sfr1) are presented. Analytical ultracentrifugation of the purified samples showed that Swi5 and the protein complex exist as tetramers and heterodimers in solution, respectively. Swi5 was crystallized in two forms belonging to space groups C2 and R3 and the crystals diffracted to 2.7 A resolution. Swi5-DeltaN180Sfr1 was crystallized in space group P2(1)2(1)2 and the crystals diffracted to 2.3 A resolution. The crystals of Swi5 and Swi5-DeltaN180Sfr1 are likely to contain one tetramer and two heterodimers in the asymmetric unit, respectively.

DNA Binding by the Mis4Scc2 Loader Promotes Topological DNA Entrapment by the Cohesin Ring.

Cohesin, a critical mediator of genome organization including sister chromatid cohesion, is a ring-shaped multi-subunit ATPase that topologically embraces DNA. Its loading and function on chromosomes require the Scc2-Scc4 loader. Using biochemical reconstitution, we show here that the ability of the loader to bind DNA plays a critical role in promoting cohesin loading. Two distinct sites within the Mis4Scc2 subunit are found to cooperatively bind DNA. Mis4Scc2 initially forms a tertiary complex with cohesin on DNA and promotes subsequent topological DNA entrapment by cohesin through its DNA binding activity, a process that requires an additional DNA binding surface provided by Psm3Smc3, the ATPase domain of cohesin. Furthermore, we show that mutations in the two DNA binding sites of Mis4 impair the chromosomal loading of cohesin. These observations demonstrate the physiological importance of DNA binding by the loader and provide mechanistic insights into the process of topological cohesin loading.

Cooperative interactions facilitate stimulation of Rad51 by the Swi5-Sfr1 auxiliary factor complex.

Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. We present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a conserved auxiliary factor. Two distinct sites within the intrinsically disordered N-terminus of Sfr1 (Sfr1N) were found to cooperatively bind Rad51. Deletion of this domain impaired Rad51 stimulation in vitro and rendered cells sensitive to DNA damage. By contrast, amino acid-substitution mutants, which had comparable biochemical defects, could promote DNA repair, suggesting that Sfr1N has another role in addition to Rad51 binding. Unexpectedly, the DNA repair observed in these mutants was dependent on Rad55-Rad57, another auxiliary factor complex hitherto thought to function independently of Swi5-Sfr1. When combined with the finding that they form a higher-order complex, our results imply that Swi5-Sfr1 and Rad55-Rad57 can collaboratively stimulate Rad51 in Schizosaccharomyces pombe.

The DNA within cells contains the instructions necessary for life and it must be carefully maintained. DNA is constantly being damaged by radiation and other factors so cells have evolved an arsenal of mechanisms that repair this damage. An enzyme called Rad51 drives one such DNA repair process known as homologous recombination. A pair of regulatory proteins known as the Swi5-Sfr1 complex binds to Rad51 and activates it. The complex can be thought of as containing two modules with distinct roles: one comprising the first half of the Sfr1 protein and that is capable of binding to Rad51, and a second consisting of the rest of Sfr1 bound to Swi5, which is responsible for activating Rad51. Here, Argunhan, Sakakura et al. used genetic and biochemical approaches to study how this first module, known as "Sfr1N", interacts with Rad51 in a microbe known as fission yeast. The experiments showed that both modules of Swi5-Sfr1 were important for Rad51 to drive homologous recombination. Swi5-Sfr1 complexes carrying mutations in the region of Sfr1N that binds to Rad51 were unable to activate Rad51 in a test tube. However, fission yeast cells containing the same mutations were able to repair their DNA without problems. This was due to the presence of another pair of proteins known as the Rad55-Rad57 complex that also bound to Swi5-Sfr1. The findings of Argunhan, Sakakura et al. suggest that the Swi5-Sfr1 and Rad55-Rad57 complexes work together to activate Rad51. Many genetically inherited diseases and cancers have been linked to mutations in DNA repair proteins. The fundamental mechanisms of DNA repair are very similar from yeast to humans and other animals, therefore, understanding the details of DNA repair in yeast may ultimately benefit human health in the future.

Real-time tracking reveals catalytic roles for the two DNA binding sites of Rad51.

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases.