A comparative analysis of Dmc1 and Rad51 nucleoprotein filaments

The eukaryotic RecA homologs Rad51 and Dmc1 are essential for strand exchange between homologous chromosomes during meiosis. All members of the RecA family of recombinases polymerize on DNA to form helical nucleoprotein filaments, which is the active form of the protein. Here we compare the filament structures of the Rad51 and Dmc1 proteins from both human and budding yeast. Previous studies of Dmc1 filaments suggested that they might be structurally distinct from filaments of other members of the RecA family, including Rad51. The data presented here indicate that Rad51 and Dmc1 filaments are essentially identical with respect to several structural parameters, including persistence length, helical pitch, filament diameter, DNA base pairs per helical turn and helical handedness. These data, together with previous studies demonstrating similar in vitro recombinase activity for Dmc1 and Rad51, support the view that differences in the meiotic function of Rad51 and Dmc1 are more likely to result from the influence of distinct sets of accessory proteins than from intrinsic differences in filament structure.     

The uvsX protein of bacteriophage T4 arranges single-stranded and double-stranded DNA into similar helical nucleoprotein filaments

The bacteriophage T4 uvsX gene codes for a DNA-binding protein that is important for genetic recombination in T4-infected cells. This protein is a DNA-dependent ATPase that resembles the Escherichia coli recA protein in many of its properties. We have examined the binding of purified uvsX protein to single-stranded DNA (ssDNA) and to double-stranded DNA (dsDNA) using electron microscopy to visualize the complexes that are formed and double label analysis to measure their protein content. We find that the uvsX protein binds cooperatively to dsDNA, forming filaments 14 nm in diameter with an apparently helical axial repeat of 12 nm. Each repeat contains about 42 base pairs and 9-12 uvsX protein monomers. In solutions containing Mg2+, the uvsX protein also binds cooperatively to ssDNA. The filaments that result are 14 nm in diameter, show a 12-nm axial repeat, and they are nearly identical in appearance to the filaments that contain dsDNA. In the filaments formed along ssDNA, each axial repeat contains about 49 DNA bases and 9-12 uvsX monomers. Both the filaments formed on the ssDNA and dsDNA show a strong tendency to align side-by-side. T4 gene 32 protein also binds cooperatively to ssDNA and interacts both physically and functionally with uvsX protein. However, when gene 32 and uvsX proteins were added to ssDNA together, no interaction between the two proteins was detected.     

Calcium ion promotes yeast Dmc1 activity via formation of long and fine helical filaments with single-stranded DNA

Dmc1 is specifically required for homologous recombination during meiosis. Here we report that the calcium ion enabled Dmc1 from budding yeast to form regular helical filaments on single-stranded DNA (ssDNA) and activate its strand assimilation activity. Relative to magnesium, calcium increased the affinity of Dmc1 for ATP and but reduces its DNA-dependent ATPase activity. These effects, together with previous studies of other RecA-like recombinases, support the view that ATP binding to Dmc1 protomers is required for functional filament structure. The helical pitch of the Saccharomyces cerevisiae Dmc1-ssDNA helical filament was estimated to be 13.4 +/- 2.5 nm. Analysis of apparently "complete" Dmc1-ssDNA filaments indicated a stoichiometry of 24 +/- 2 nucleotides per turn of the Dmc1 helix. This finding suggests that the number or protomers per helical turn and/or the number of nucleotides bound per Dmc1 protomer differs from that reported previously for Rad51 and RecA filaments. Our data support the view that the active form of Dmc1 protein is a helical filament rather than a ring. We speculate that Ca(2+) plays a significant role in regulating meiotic recombination.     

Stimulation of fission yeast and mouse Hop2-Mnd1 of the Dmc1 and Rad51 recombinases

Genetic analysis of fission yeast suggests a role for the spHop2–Mnd1 proteins in the Rad51 and Dmc1-dependent meiotic recombination pathways. In order to gain biochemical insights into this process, we purified Schizosaccharomyces pombe Hop2-Mnd1 to homogeneity. spHop2 and spMnd1 interact by co-immunoprecipitation and two-hybrid analysis. Electron microscopy reveals that S. pombe Hop2–Mnd1 binds single-strand DNA ends of 3′-tailed DNA. Interestingly, spHop2-Mnd1 promotes the renaturation of complementary single-strand DNA and catalyses strand exchange reactions with short oligonucleotides. Importantly, we show that spHop2-Mnd1 stimulates spDmc1-dependent strand exchange and strand invasion. Ca²⁺ alleviate the requirement for the order of addition of the proteins on DNA. We also demonstrate that while spHop2-Mnd1 affects spDmc1 specifically, mHop2 or mHop2-Mnd1 stimulates both the hRad51 and hDmc1 recombinases in strand exchange assays. Thus, our results suggest a crucial role for S. pombe and mouse Hop2-Mnd1 in homologous pairing and strand exchange and reveal evolutionary divergence in their specificity for the Dmc1 and Rad51 recombinases.     

Influence of DNA repair defects (rad1, rad52) on nitrogen mustard mutagenesis in yeast.

Summary Nitrogen mustard (HN2) mutagenesis of a plasmid-borne copy of the Saccharomyces cerevisiae SUP4-o gene was examined in a repair-proficient yeast strain and isogenic derivatives defective for excision (radl) or DNA double-strand break (rad52) repair. The excision repair deficiency sensitized the cells to killing by HN2 and abolished mutation induction. Inactivation of RAD52 had no influence on the lethality of HN2 treatment but diminished the induced mutation frequency by 50% at all doses tested. DNA sequence analysis of HN2-induced SUP4-o mutations suggested that RAD52 contributed to the production of basepair substitutions at G·C sites. The rad52 defect appeared to alter the distribution of G·C A·T transitions in SUP4-o relative to the distribution for the wild-type strain. This difference did not seem to be due to an effect of RAD52 on the relative fractions of HN2-induced transitions at localized (flanked by A·T pairs) or contiguous (flanked by at least one G·C pair) G·C sites but instead to an influence on the strand specificity of HN2 mutagenesis. In the repair-proficient strain, the transitions showed a small bias for sites having the guanine on the transcribed strand and this preference was eliminated by inactivation of RAD52.     

The stretched DNA geometry of recombination and repair nucleoprotein filaments

The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes. To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA x tsDNA complex), has been elucidated by classical methods. Here we review the systematic characterization of the helical geometries of the three DNA strands of the RecA x tsDNA complex using fluorescence resonance energy transfer (FRET) under physiologically relevant solution conditions. Measurements of the helical parameters for the RecA x tsDNA complex are consistent with the hypothesis that this complex is a late, poststrand-exchange intermediate with the outgoing strand shifted by about three base pairs with respect to its registry with the incoming and complementary strands. All three strands in the RecA x tsDNA complex adopt extended and unwound conformations similar to those of RecA-bound ssDNA and dsDNA.     

Real-time measurements of the nucleation, growth and dissociation of single Rad51-DNA nucleoprotein filaments

Human Rad51 (hRad51), the protein central to DNA pairing and strand exchange during homologous recombination, polymerizes on DNA to form nucleoprotein filaments. By making use of magnetic tweezers to manipulate individual DNA molecules, we measured the nucleation and growth of hRad51 nucleoprotein filaments, and their subsequent disassembly in real time. The dependence of the initial polymerization rate upon the concentration of hRad51 suggests that the rate-limiting step is the formation of a nucleus involving 5.5 ± 1.5 hRad51 monomers, corresponding to one helical turn of the hRad51 nucleoprotein filament. Polymerization is highly cooperative (i.e. a nucleation-limited reaction) at low concentrations and less cooperative (a growth-limited reaction) at high concentrations of the protein. We show that the observed preference of hRad51 to form nucleoprotein filaments on double-stranded DNA rather than on single-stranded DNA is due to the fact that it depolymerizes much faster from ssDNA than from dsDNA: indeed, hRad51 polymerizes faster on ssDNA than on dsDNA. Hydrolysis of ATP by hRad51 does not correlate with its dissociation from dsDNA. This suggests that hRad51 does not depolymerize rapidly from dsDNA after strand exchange but stays bound to the heteroduplex, highlighting the importance of partner proteins to facilitate hRad51 depolymerization from dsDNA.     

Fission yeast Pot1 and RecQ helicase are required for efficient chromosome segregation

Pot1 is a single-stranded telomere-binding protein that is conserved from fission yeast to mammals. Deletion of Schizosaccharomyces pombe pot1(+) causes immediate telomere loss. S. pombe Rqh1 is a homolog of the human RecQ helicase WRN, which plays essential roles in the maintenance of genomic stability. Here, we demonstrate that a pot1Δ rqh1-hd (helicase-dead) double mutant maintains telomeres that are dependent on Rad51-mediated homologous recombination. Interestingly, the pot1Δ rqh1-hd double mutant displays a "cut" (cell untimely torn) phenotype and is sensitive to the antimicrotubule drug thiabendazole (TBZ). Moreover, the chromosome ends of the double mutant do not enter the pulsed-field electrophoresis gel. These results suggest that the entangled chromosome ends in the pot1Δ rqh1-hd double mutant inhibit chromosome segregation, signifying that Pot1 and Rqh1 are required for efficient chromosome segregation. We also found that POT1 knockdown, WRN-deficient human cells are sensitive to the antimicrotubule drug vinblastine, implying that some of the functions of S. pombe Pot1 and Rqh1 may be conserved in their respective human counterparts POT1 and WRN.     

Fission yeast rad17: a homologue of budding yeast RAD24 that shares regions of sequence similarity with DNA polymerase accessory proteins.

Following DNA damage or a block to DNA synthesis, checkpoint pathways act to arrest mitosis and prevent the attempted segregation of damaged or unreplicated DNA. The rad17 locus of Schizosaccharomyces pombe is one of seven known radiation-sensitive (rad) loci which are absolutely required to prevent mitosis following DNA damage in fission yeast. Six of these (rad1, rad3, rad9, rad17, rad26 and hus1) are also required for the checkpoint which prevents mitosis from occurring before DNA replication is complete. We report here that the predicted rad17 gene product is a basic hydrophilic protein of 606 amino acids which contains five domains with sequence homology to replication factor C (RF-C)/activator 1 subunits. Western analysis and fusion with Green Fluorescent Protein indicate that the abundance and electrophoretic mobility of Rad17 is not significantly modified following a block to DNA synthesis or following DNA damage, and that Rad17 is localized in the nucleus. Rad17 function is not essential for growth, but is required for the function of the DNA structure-dependent checkpoints. Site-directed mutagenesis has been used to demonstrate the biological significance of the RF-C/activator 1-related domains. These studies have also defined an element of the radiation sensitivity caused by loss of Rad17 function which is not associated with the radiation-induced G2 arrest defect seen in the rad17.d null mutant cells.     

Fission yeast Stn1 maintains stability of repetitive DNA at subtelomere and ribosomal DNA regions

Telomere binding protein Stn1 forms the CST (Cdc13/CTC1-STN1-TEN1) complex in budding yeast and mammals. Likewise, fission yeast Stn1 and Ten1 form a complex indispensable for telomere protection. We have previously reported that stn1-1, a high-temperature sensitive mutant, rapidly loses telomere DNA at the restrictive temperature due to frequent failure of replication fork progression at telomeres and subtelomeres, both containing repetitive sequences. It is unclear, however, whether Stn1 is required for maintaining other repetitive DNAs such as ribosomal DNA. In this study, we have demonstrated that stn1-1 cells, even when grown at the permissive temperature, exhibited dynamic rearrangements in the telomere-proximal regions of subtelomere and ribosomal DNA repeats. Furthermore, Rad52 and γH2A accumulation was observed at ribosomal DNA repeats in the stn1-1 mutant. The phenotypes exhibited by the stn1-1 allele were largely suppressed in the absence of Reb1, a replication fork barrier-forming protein, suggesting that Stn1 is involved in the maintenance of the arrested replication forks. Collectively, we propose that Stn1 maintains the stability of repetitive DNAs at subtelomeres and rDNA regions.     

Fission yeast Swi1-Swi3 complex facilitates DNA binding of Mrc1

Replication fork protection complex Swi1-Swi3 and replication checkpoint mediator Mrc1 are required for maintenance of replication fork integrity during the course of DNA replication in the fission yeast Schizosaccharomyces pombe. These proteins play crucial roles in stabilizing stalled forks and activating replication checkpoint signaling pathways. Although they are conserved replication fork components, precise biochemical roles of these proteins are not known. Here we purified Mrc1 and Swi1-Swi3 proteins and show that these proteins bind to DNA independently but synergistically in vitro. Mrc1 binds preferentially to arrested fork or D-loop-like structures, although the affinity is relatively low, whereas the Swi1-Swi3 complex binds to double-stranded DNA with higher affinity. In the presence of a low concentration of Swi1-Swi3, Mrc1 generates a novel ternary complex and binds to various types of DNA with higher affinity. Moreover, purified Mrc1 and Swi1-Swi3 physically interact with each other, and this interaction is lost by mutations in the known DNA binding domain of Mrc1 (K235E,K236E). The interaction is also lost in a mutant form of Swi1 (E662K) that is specifically defective in polar fork arrest at a site called RTS1 and causes sensitivity to genotoxic agents, although the DNA binding affinity of Swi1-Swi3 is not affected by this mutation. As expected, the synergistic effect of the Swi1-Swi3 on DNA binding of Mrc1 is also lost by these mutations affecting the interaction between Mrc1 and Swi1-Swi3. Our results reveal an aspect of molecular interactions that may play an important role in replication pausing and fork stabilization.     

The Rad51 and Dmc1 recombinases: a non-identical twin relationship

A double-strand break in genomic DNA that remains unrepaired can be lethal for a cell. Indeed, the integrity of the genome is paramount for survival. It is therefore surprising that some cells deliberately introduce double-strand breaks at certain times during their life cycle. Why might they do this? What are the benefits? How are these breaks repaired? The answers to these questions lie in understanding the basis of meiotic recombination, the process that leads to genetic variation. This review summarizes the key roles played by the two recombinases, Dmc1 and Rad51, in the faithful repair of DNA breaks.     

Indistinguishable Landscapes of Meiotic DNA Breaks in rad50+ and rad50S Strains of Fission Yeast Revealed by a Novel rad50+ Recombination Intermediate

The fission yeast Schizosaccharomyces pombe Rec12 protein, the homolog of Spo11 in other organisms, initiates meiotic recombination by creating DNA double-strand breaks (DSBs) and becoming covalently linked to the DNA ends of the break. This protein–DNA linkage has previously been detected only in mutants such as rad50S in which break repair is impeded and DSBs accumulate. In the budding yeast Saccharomyces cerevisiae, the DSB distribution in a rad50S mutant is markedly different from that in wild-type (RAD50) meiosis, and it was suggested that this might also be true for other organisms. Here, we show that we can detect Rec12-DNA linkages in Sc. pombe rad50⁺ cells, which are proficient for DSB repair. In contrast to the results from Sa. cerevisiae, genome-wide microarray analysis of Rec12-DNA reveals indistinguishable meiotic DSB distributions in rad50⁺ and rad50S strains of Sc. pombe. These results confirm our earlier findings describing the occurrence of widely spaced DSBs primarily in large intergenic regions of DNA and demonstrate the relevance and usefulness of fission yeast studies employing rad50S. We propose that the differential behavior of rad50S strains reflects a major difference in DSB regulation between the two species—specifically, the requirement for the Rad50-containing complex for DSB formation in budding yeast but not in fission yeast. Use of rad50S and related mutations may be a useful method for DSB analysis in other species.     

Fission yeast Swi5 protein, a novel DNA recombination mediator

The Schizosaccharomyces pombe Swi5 protein forms two distinct protein complexes, Swi5-Sfr1 and Swi5-Swi2, each of which plays an important role in the related but functionally distinct processes of homologous recombination and mating-type switching, respectively. The Swi5-Sfr1 mediator complex has been shown to associate with the two RecA-like recombinases, Rhp51 (spRad51) and Dmc1, and to stimulate in vitro DNA strand exchange reactions mediated by these proteins. Genetic analysis indicates that Swi5-Sfr1 works independently of another mediator complex, Rhp55-Rhp57, during Rhp51-dependent recombinational repair. In addition, mutations affecting the two mediators generate distinct repair spectra of HO endonuclease-induced DNA double strand breaks, suggesting that these recombination mediators differently regulate recombination outcomes in an independent manner.     

Interaction of human rad51 recombination protein with single-stranded DNA binding protein, RPA.

Replication protein A (RPA), a heterotrimeric single-stranded DNA binding protein, is required for recombination, and stimulates homologous pairing and DNA strand exchange promoted in vitro by human recombination protein HsRad51. Co-immunoprecipitation revealed that purified RPA interacts physically with HsRad51, as well as with HsDmc1, the homolog that is expressed specifically in meiosis. The interaction with HsRad51 was mediated by the 70 kDa subunit of RPA, and according to experiments with deletion mutants, this interaction required amino acid residues 169-326. In exponentially growing mammalian cells, 22% of nuclei showed foci of RPA protein and 1-2% showed foci of Rad51. After gamma-irradiation, the percentage of cells with RPA foci increased to approximately 50%, and those with Rad51 foci to 30%. All of the cells with foci of Rad51 had foci of RPA, and in those cells the two proteins co-localized in a high fraction of foci. The interactions of human RPA with Rad51, replication proteins and DNA are suited to the linking of recombination to replication.     

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.     

Stable and efficient cassette exchange under non-selectable conditions by combined use of two site-specific recombinases

Work of the last decade has proven the 'one gene- one product-one function' hypothesis an oversimplification. To further unravel the emerging 'one gene-multiple products-even more functions' concept, new methods (such as subtle knock-in and tightly regulated conditional mutations) for the analysis of gene function in health and disease are required. Another class of improvements (such as tetraploid fusion and cassette exchange) addresses the efficiency with which targeted mutant strains can be generated. Recombinase-mediated cassette exchange (RMCE), which in theory is well suited for the rapid generation of multiple alleles of a given locus, is hampered by its low efficiency in the absence of selection and, especially in vivo, by the promiscuity of the participating recombinase recognition sites. Here we present a novel approach which circumvents this problem by the use of two independent recombinase systems. The strategy, which uses loxP on one and FRT on the other side of the cassette together with a Cre/Flpe expression vector, prevents excisive events and results in higher rates of cassette integration without selection than previously described. This method has a huge potential for the generation of allelic series in embryonic stem cells and, importantly, in pre-implantation embryos in vivo.     

Robust and efficient synthetic method for forming DNA microarrays

The field of DNA microarray technology has necessitated the cooperative efforts of interdisciplinary scientific teams to achieve its primary goal of rapidly measuring global gene expression patterns. A collaborative effort was established to produce a chemically reactive surface on glass slide substrates to which unmodified DNA will covalently bind for improvement of cDNA microarray technology. Using the p-aminophenyl trimethoxysilane (ATMS)/diazotization chemistry that was developed, microarrays were fabricated and analyzed. This immobilization method produced uniform spots containing equivalent or greater amounts of DNA than commercially available immobilization techniques. In addition, hybridization analyses of microarrays made with ATMS/diazotization chemistry showed very sensitive detection of the target sequence, two to three orders of magnitude more sensitive than the commercial chemistries. Repeated stripping and re-hybridization of these slides showed that DNA loss was minimal, allowing multiple rounds of hybridization. Thus, the ATMS/diazotization chemistry facilitated covalent binding of unmodified DNA, and the reusable microarrays that were produced showed enhanced levels of hybridization and very low background fluorescence.     

Fission yeast Swi5/Sfr1 and Rhp55/Rhp57 differentially regulate Rhp51-dependent recombination outcomes

Several accessory proteins referred to as mediators are required for the full activity of the Rad51 (Rhp51 in fission yeast) recombinase. In this study, we analyzed in vivo functions of the recently discovered Swi5/Sfr1 complex from fission yeast. In normally growing cells, the Swi5-GFP protein localizes to the nucleus, where it forms a diffuse nuclear staining pattern with a few distinct foci. These spontaneous foci do not form in swi2Delta mutants. Upon UV irradiation, Swi5 focus formation is induced in swi2Delta mutants, a response that depends on Sfr1 function, and Sfr1 also forms foci that colocalize with damage-induced Rhp51 foci. The number of UV-induced Rhp51 foci is partially reduced in swi5Delta and rhp57Delta mutants and completely abolished in an swi5Delta rhp57Delta double mutant. An assay for products generated by HO endonuclease-induced DNA double-strand breaks (DSBs) reveals that Rhp51 and Rhp57, but not Swi5/Sfr1, are essential for crossover production. These results suggest that Swi5/Sfr1 functions as an Rhp51 mediator but processes DSBs in a manner different from that of the Rhp55/57 mediator.     

Significance of ligand interactions involving Hop2-Mnd1 and the RAD51 and DMC1 recombinases in homologous DNA repair and XX ovarian dysgenesis

The evolutionarily conserved Hop2-Mnd1 complex is a key cofactor for the meiosis-specific recombinase Dmc1. However, emerging evidence has revealed that Hop2-Mnd1 is expressed in somatic tissues, primary human fibroblasts and cell lines, and that it functions in conjunction with the Rad51 recombinase to repair damaged telomeres via the alternate lengthening of telomeres mechanism. Here, we reveal how distinct DNA-binding activities of Hop2-Mnd1 mediate the stabilization of the RAD51-ssDNA presynaptic filament or stimulate the homologous DNA pairing reaction. We have also endeavored to define the interface that governs the assembly of the higher order complex of Hop2-Mnd1 with RAD51. Unexpectedly, we find that ATP enhances the interaction between Hop2-Mnd1 and RAD51, and that both Hop2 and Mnd1 are involved in RAD51 interaction via their C-terminal regions. Importantly, mutations introduced into these Hop2 and Mnd1 domains, including the HOP2 p.del201Glu mutation present in a patient of XX ovarian dysgenesis, diminish the association and functional synergy of Hop2-Mnd1 with both RAD51 and DMC1. Our findings help delineate the intricate manner in which Hop2-Mnd1 engages and functions with RAD51 and DMC1 in mammalian cells and speak to the possible cause of XX ovarian dysgenesis.