Human DMC1 Research Articles

The role of bivalent ions in the regulation of D-loop extension mediated by DMC1 during meiotic recombination

During meiosis, programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination. DMC1, a conserved recombinase, plays a central role in this process. DMC1 promotes DNA strand exchange between homologous chromosomes, thus creating the physical linkage between them. Its function is regulated not only by several accessory proteins but also by bivalent ions. Here, we show that whereas calcium ions in the presence of ATP cause a conformational change within DMC1, stimulating its DNA binding and D-loop formation, they inhibit the extension of the invading strand within the D-loop. Based on structural studies, we have generated mutants of two highly conserved amino acids- E162 and D317- in human DMC1, which are deficient in calcium regulation. Invivo studies of their yeast homologues further showed that they exhibit severe defects in meiosis, thus emphasizing the importance of calcium ions in the regulation of DMC1 function and meiotic recombination.

Mechanisms of distinctive mismatch tolerance between Rad51 and Dmc1 in homologous recombination.

Homologous recombination (HR) is a primary DNA double-strand breaks (DSBs) repair mechanism. The recombinases Rad51 and Dmc1 are highly conserved in the RecA family; Rad51 is mainly responsible for DNA repair in somatic cells during mitosis while Dmc1 only works during meiosis in germ cells. This spatiotemporal difference is probably due to their distinctive mismatch tolerance during HR: Rad51 does not permit HR in the presence of mismatches, whereas Dmc1 can tolerate certain mismatches. Here, the cryo-EM structures of Rad51–DNA and Dmc1–DNA complexes revealed that the major conformational differences between these two proteins are located in their Loop2 regions, which contain invading single-stranded DNA (ssDNA) binding residues and double-stranded DNA (dsDNA) complementary strand binding residues, stabilizing ssDNA and dsDNA in presynaptic and postsynaptic complexes, respectively. By combining molecular dynamic simulation and single-molecule FRET assays, we identified that V273 and D274 in the Loop2 region of human RAD51 (hRAD51), corresponding to P274 and G275 of human DMC1 (hDMC1), are the key residues regulating mismatch tolerance during strand exchange in HR. This HR accuracy control mechanism provides mechanistic insights into the specific roles of Rad51 and Dmc1 in DNA double-strand break repair and may shed light on the regulatory mechanism of genetic recombination in mitosis and meiosis.

Identification of fidelity-governing factors in human recombinases DMC1 and RAD51 from cryo-EM structures

Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open “triplet gate” for mismatch tolerance. In support, DMC1-Q244M displayed marked increase in DNA dynamics, leading to unobservable DNA map. MD simulation showed highly dispersive mismatched DNA ensemble in RAD51 but well-converged DNA in DMC1 and RAD51-V273P/D274G. Replacing Loop 1 or Loop 2 residues in DMC1 with RAD51 counterparts enhanced DMC1 fidelity, while reciprocal mutations in RAD51 attenuated its fidelity. Our results show that three Loop 1/Loop 2 residues jointly enact contrasting fidelities of DNA recombinases.

In Vitro Assay for Plasmid Length DNA Strand Exchange by Human DMC1.

Meiosis is a specialized cell division that generates gametes. Meiotic recombination is essential not only to generate diversity in offspring, but also to hold homologous chromosomes together through chiasma allowing proper chromosome segregation. This process requires the meiosis-specific recombinase, DMC1. DMC1 facilitates the search for homology between the homologous chromosomes and is followed by DNA strand invasion and strand exchange to produce a linkage between the two homologous chromosomes. The development of biochemical in vitro assays and the purification of the requisite proteins factors has led to a better understanding of the molecular mechanisms of meiotic homologous recombination. In this chapter, a detailed in vitro assay to examine DNA strand exchange over 5000 bases of DNA catalyzed by human DMC1 is described. This method has proved to be valuable for examining the catalytic potential of hDMC1 and delineating the functional interaction with other HR factors.

Stabilization of the Human DMC1 Nucleoprotein Filament.

The meiosis-specific recombinase, DMC1, is important for the generation of haploids during meiosis. DMC1 forms a helical nucleoprotein filament on ssDNA overhangs located at the processed double-stranded DNA break. The DMC1 filament performs a search for homology in homologous chromosome. Once homology is located, the DMC1 filament strand invades the homologous chromosome forming a displacement loop (D-loop). These connections are needed for accurate segregation to occur later in meiosis. Because DMC1 requires numerous accessory factors and specific ionic conditions to participate in this DNA repair process, in vitro assays were developed to understand how these accessory factors influence the biochemical properties of hDMC1. This chapter describes a method that can be used to investigate the stability of the human DMC1 nucleoprotein filament under various conditions and provides insight into an important early stage in DNA double-strand break repair by homologous recombination during meiosis.

BRCA2 regulates DMC1-mediated recombination through the BRC repeats

In somatic cells, BRCA2 is needed for RAD51-mediated homologous recombination. The meiosis-specific DNA strand exchange protein, DMC1, promotes the formation of DNA strand invasion products (joint molecules) between homologous molecules in a fashion similar to RAD51. BRCA2 interacts directly with both human RAD51 and DMC1; in the case of RAD51, this interaction results in stimulation of RAD51-promoted DNA strand exchange. However, for DMC1, little is known regarding the basis and functional consequences of its interaction with BRCA2. Here we report that human DMC1 interacts directly with each of the BRC repeats of BRCA2, albeit most tightly with repeats 1-3 and 6-8. However, BRC1-3 bind with higher affinity to RAD51 than to DMC1, whereas BRC6-8 bind with higher affinity to DMC1, providing potential spatial organization to nascent filament formation. With the exception of BRC4, each BRC repeat stimulates joint molecule formation by DMC1. The basis for this stimulation is an enhancement of DMC1-ssDNA complex formation by the stimulatory BRC repeats. Lastly, we demonstrate that full-length BRCA2 protein stimulates DMC1-mediated DNA strand exchange between RPA-ssDNA complexes and duplex DNA, thus identifying BRCA2 as a mediator of DMC1 recombination function. Collectively, our results suggest unique and specialized functions for the BRC motifs of BRCA2 in promoting homologous recombination in meiotic and mitotic cells.

Functional Relationship of ATP Hydrolysis, Presynaptic Filament Stability, and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1

DMC1 and RAD51 are conserved recombinases that catalyze homologous recombination. DMC1 and RAD51 share similar properties in DNA binding, DNA-stimulated ATP hydrolysis, and catalysis of homologous DNA strand exchange. A large body of evidence indicates that attenuation of ATP hydrolysis leads to stabilization of the RAD51-ssDNA presynaptic filament and enhancement of DNA strand exchange. However, the functional relationship of ATPase activity, presynaptic filament stability, and DMC1-mediated homologous DNA strand exchange has remained largely unexplored. To address this important question, we have constructed several mutant variants of human DMC1 and characterized them biochemically to gain mechanistic insights. Two mutations, K132R and D223N, that change key residues in the Walker A and B nucleotide-binding motifs ablate ATP binding and render DMC1 inactive. On the other hand, the nucleotide-binding cap D317K mutant binds ATP normally but shows significantly attenuated ATPase activity and, accordingly, forms a highly stable presynaptic filament. Surprisingly, unlike RAD51, presynaptic filament stabilization achieved via ATP hydrolysis attenuation does not lead to any enhancement of DMC1-catalyzed homologous DNA pairing and strand exchange. This conclusion is further supported by examining wild-type DMC1 with non-hydrolyzable ATP analogues. Thus, our results reveal an important mechanistic difference between RAD51 and DMC1.

Structure of a filament of stacked octamers of human DMC1 recombinase.

Eukaryal DMC1 proteins play a central role in homologous recombination in meiosis by assembling at the sites of programmed DNA double-strand breaks and carrying out a search for allelic DNA sequences located on homologous chromatids. They are close homologs of eukaryal Rad51 and archaeal RadA proteins and are remote homologs of bacterial RecA proteins. These recombinases (also called DNA strand-exchange proteins) promote a pivotal strand-exchange reaction between homologous single-stranded and double-stranded DNA substrates. An octameric form of a truncated human DMC1 devoid of its small N-terminal domain (residues 1-83) has been crystallized. The structure of the truncated DMC1 octamer is similar to that of the previously reported full-length DMC1 octamer, which has disordered N-terminal domains. In each protomer, only the ATP cap regions (Asp317-Glu323) show a noticeable conformational difference. The truncated DMC1 octamers further stack with alternate polarity into a filament. Similar filamentous assemblies of DMC1 have been observed to form on DNA by electron microscopy.

Role of the conserved lysine within the Walker A motif of human DMC1

During meiosis, the RAD51 recombinase and its meiosis-specific homolog DMC1 mediate DNA strand exchange between homologous chromosomes. The proteins form a right-handed nucleoprotein complex on ssDNA called the presynaptic filament. In an ATP-dependent manner, the presynaptic filament searches for homology to form a physical connection with the homologous chromosome. We constructed two variants of hDMC1 altering the conserved lysine residue of the Walker A motif to arginine (hDMC1K132R) or alanine (hDMC1K132A). The hDMC1 variants were expressed in Escherichia coli and purified to near homogeneity. Both hDMC1K132R and hDMC1K132A variants were devoid of ATP hydrolysis. The hDMC1K132R variant was attenuated for ATP binding that was partially restored by the addition of either ssDNA or calcium. The hDMC1K132R variant was partially capable of homologous DNA pairing and strand exchange in the presence of calcium and protecting DNA from a nuclease, while the hDMC1K132A variant was inactive. These results suggest that the conserved lysine of the Walker A motif in hDMC1 plays a key role in ATP binding. Furthermore, the binding of calcium and ssDNA promotes a conformational change in the ATP binding pocket of hDMC1 that promotes ATP binding. Our results provide evidence that the conserved lysine in the Walker A motif of hDMC1 is critical for ATP binding which is required for presynaptic filament formation.

Genome wide cloning of maize meiotic recombinase Dmc1 and its functional structure through molecular phylogeny

The development of meiotic division and associated genetic recombination paved the way for evolutionary changes. However, the secondary and tertiary structure and functional domains of many of the proteins involved in genetic recombination have not been studied in detail. We used the human Dmc1 gene product along with secondary and tertiary domain structures of Escherichia coli RecA protein to help determine the molecular structure and function of maize Dmc1, which is required for synaptonemal complex formation and cell cycle progression. The maize recombinase Dmc1 gene was cloned and characterized, using rice Dmc1 cDNA as an orthologue. The deduced amino acid sequence was used for elaborating its 3-D structure, and functional analysis was made with the CDD software, showing significant identity of the Dmc1 gene product in Zea mays with that of Homo sapiens. Based on these results, the domains and motives of WalkerA and WalkerB as ATP binding sites, a multimer site (BRC) interface, the putative ssDNA binding L1 and L2 loops, the putative dsDNA binding helix-hairpin-helix, a polymerization motif, the subunit rotation motif, and a small N-terminal domain were proposed for maize recombinase Dmc1.

Helical Filaments of Human Dmc1 Protein on Single-Stranded DNA: A Cautionary Tale

Proteins in the RecA/Rad51/RadA family form nucleoprotein filaments on DNA that catalyze a strand exchange reaction as part of homologous genetic recombination. Because of the centrality of this system to many aspects of DNA repair, the generation of genetic diversity, and cancer when this system fails or is not properly regulated, these filaments have been the object of many biochemical and biophysical studies. A recent paper has argued that the human Dmc1 protein, a meiotic homolog of bacterial RecA and human Rad51, forms filaments on single-stranded DNA with ∼9 subunits per turn in contrast to the filaments formed on double-stranded DNA with ∼6.4 subunits per turn and that the stoichiometry of DNA binding is different between these two filaments. We show using scanning transmission electron microscopy that the Dmc1 filament formed on single-stranded DNA has a mass per unit length expected from ∼6.5 subunits per turn. More generally, we show how ambiguities in helical symmetry determination can generate incorrect solutions and why one sometimes must use other techniques, such as biochemistry, metal shadowing, or scanning transmission electron microscopy, to resolve these ambiguities. While three-dimensional reconstruction of helical filaments from EM images is a powerful tool, the intrinsic ambiguities that may be present with limited resolution are not sufficiently appreciated.

Structure of the hDmc1-ssDNA Filament Reveals the Principles of Its Architecture

In eukaryotes, meiotic recombination is a major source of genetic diversity, but its defects in humans lead to abnormalities such as Down's, Klinefelter's and other syndromes. Human Dmc1 (hDmc1), a RecA/Rad51 homologue, is a recombinase that plays a crucial role in faithful chromosome segregation during meiosis. The initial step of homologous recombination occurs when hDmc1 forms a filament on single-stranded (ss) DNA. However the structure of this presynaptic complex filament for hDmc1 remains unknown. To compare hDmc1-ssDNA complexes to those known for the RecA/Rad51 family we have obtained electron microscopy (EM) structures of hDmc1-ssDNA nucleoprotein filaments using single particle approach. The EM maps were analysed by docking crystal structures of Dmc1, Rad51, RadA, RecA and DNA. To fully characterise hDmc1-DNA complexes we have analysed their organisation in the presence of Ca2+, Mg2+, ATP, AMP-PNP, ssDNA and dsDNA. The 3D EM structures of the hDmc1-ssDNA filaments allowed us to elucidate the principles of their internal architecture. Similar to the RecA/Rad51 family, hDmc1 forms helical filaments on ssDNA in two states: extended (active) and compressed (inactive). However, in contrast to the RecA/Rad51 family, and the recently reported structure of hDmc1-double stranded (ds) DNA nucleoprotein filaments, the extended (active) state of the hDmc1 filament formed on ssDNA has nine protomers per helical turn, instead of the conventional six, resulting in one protomer covering two nucleotides instead of three. The control reconstruction of the hDmc1-dsDNA filament revealed 6.4 protein subunits per helical turn indicating that the filament organisation varies depending on the DNA templates. Our structural analysis has also revealed that the N-terminal domain of hDmc1 accomplishes its important role in complex formation through domain swapping between adjacent protomers, thus providing a mechanistic basis for coordinated action of hDmc1 protomers during meiotic recombination.

Reversibility, Equilibration, and Fidelity of Strand Exchange Reaction between Short Oligonucleotides Promoted by RecA Protein from Escherichia coli and Human Rad51 and Dmc1 Proteins

We demonstrate the reversibility of RecA-promoted strand exchange reaction between short oligonucleotides in the presence of adenosine 5'-O-(thiotriphosphate). The reverse reaction proceeds without the dissociation of RecA from DNA. The reaction reaches equilibrium and its yield depends on the homology between the reaction substrates. We estimate the tolerance of the RecA-promoted strand exchange to individual base substitutions for a comprehensive set of possible base combinations in a selected position along oligonucleotide substrates for strand exchange and find, in agreement with previously reported estimations, that this tolerance is higher than in the case of free DNA. It is demonstrated that the short oligonucleotide-based approach can be applied to the human recombinases Rad51 and Dmc1 when strand exchange is performed in the presence of calcium ions and ATP. Remarkably, despite the commonly held belief that the eukaryotic recombinases have an inherently lower strand exchange activity, in our system their efficiencies in strand exchange are comparable with that of RecA. Under our experimental conditions, the human recombinases exhibit a significantly higher tolerance to interruptions of homology due to point base substitutions than RecA. Finding conditions where a chemical reaction is reversible and reaches equilibrium is critically important for its thermodynamically correct description. We believe that the experimental system described here will substantially facilitate further studies on different aspects of the mechanisms of homologous recombination.

Biochemical analysis of the human DMC1-I37N polymorphism

The DMC1 protein, a meiosis-specific DNA recombinase, promotes homologous pairing and strand exchange. The I37N single nucleotide polymorphism of the human DMC1 protein was reported as a result of human genome sequencing projects. In this study, we purified the human DMC1-I37N variant, as a recombinant protein. The DMC1 protein is known to require DNA for efficient ATP hydrolysis. By contrast, the DMC1-I37N variant efficiently hydrolyzed ATP in the absence of DNA. Like the conventional DMC1 protein, the DMC1-I37N variant promoted strand exchange, but it required a high Ca2+ concentration (4-8 mm), a condition that inactivates the strand-exchange activity of the conventional DMC1 protein. These biochemical differences between the DMC1 and DMC1-I37N proteins suggest that the DMC1-I37N polymorphism may be a source of improper meiotic recombination, causing meiotic defects in humans.

Filament formation and robust strand exchange activities of the rice DMC1A and DMC1B proteins

The DMC1 protein, a meiosis-specific DNA recombinase, catalyzes strand exchange between homologous chromosomes. In rice, two Dmc1 genes, Dmc1A and Dmc1B, have been reported. Although the Oryza sativa DMC1A protein has been partially characterized, however the biochemical properties of the DMC1B protein have not been defined. In the present study, we expressed the Oryza sativa DMC1A and DMC1B proteins in bacteria and purified them. The purified DMC1A and DMC1B proteins formed helical filaments along single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and promoted robust strand exchange between ssDNA and dsDNA over five thousand base pairs in the presence of RPA, as a co-factor. The DMC1A and DMC1B proteins also promoted strand exchange in the absence of RPA with long DNA substrates containing several thousand base pairs. In contrast, the human DMC1 protein strictly required RPA to promote strand exchange with these long DNA substrates. The strand-exchange activity of the Oryza sativa DMC1A protein was much higher than that of the DMC1B protein. Consistently, the DNA-binding activity of the DMC1A protein was higher than that of the DMC1B protein. These biochemical differences between the DMC1A and DMC1B proteins may provide important insight into their functional differences during meiosis in rice.

Structural and functional analyses of the DMC1-M200V polymorphism found in the human population

The M200V polymorphism of the human DMC1 protein, which is an essential, meiosis-specific DNA recombinase, was found in an infertile patient, raising the question of whether this homozygous human DMC1-M200V polymorphism may cause infertility by affecting the function of the human DMC1 protein. In the present study, we determined the crystal structure of the human DMC1-M200V variant in the octameric-ring form. Biochemical analyses revealed that the human DMC1-M200V variant had reduced stability, and was moderately defective in catalyzing in vitro recombination reactions. The corresponding M194V mutation introduced in the Schizosaccharomyces pombe dmc1 gene caused a significant decrease in the meiotic homologous recombination frequency. Together, these structural, biochemical and genetic results provide extensive evidence that the human DMC1-M200V mutation impairs its function, supporting the previous interpretation that this single-nucleotide polymorphism is a source of human infertility.

Role of the N-terminal domain of the human DMC1 protein in octamer formation and DNA binding. VOLUME 280 (2005) PAGES 28382-28387

Role of the N-terminal domain of the human DMC1 protein in octamer formation and DNA binding. VOLUME 280 (2005) PAGES 28382-28387

Interaction between Arabidopsis Brca2 and Its Partners Rad51, Dmc1, and Dss1

The Arabidopsis (Arabidopsis thaliana) orthologs of Brca2, a protein whose mutations are involved in breast cancer in humans, were previously shown to be essential at meiosis. In an attempt to better understand the Brca2-interacting properties, we examined four partners of the two isoforms of Brca2 identified in Arabidopsis (AtRad51, AtDmc1, and two AtDss1 isoforms). The two Brca2 and the two Dss1 isoforms are named AtBrca2(IV), AtBrca2(V), AtDss1(I), and AtDss1(V) after their chromosomal localization. We first show that both AtBrca2 proteins can interact with either AtRad51 or AtDmc1 in vitro, and that the N-terminal region of AtBrca2 is responsible for these interactions. More specifically, the BRC motifs (so called because iterated in the Brca2 protein) in Brca2 are involved in these interactions: BRC motif number 2 (BRC2) alone can interact with AtDmc1, whereas BRC motif number 4 (BRC4) recognizes AtRad51. The human Rad51 and Dmc1 proteins themselves can interact with either the complete (HsRad51) or a shorter version of AtBrca2 (HsRad51 or HsDmc1) that comprises all four BRC motifs. We also identified two Arabidopsis isoforms of Dss1, another known partner of Brca2 in other organisms. Although all four Brca2 and Dss1 proteins are much conserved, AtBrca2(IV) interacts with only one of these AtDss1 proteins, whereas AtBrca2(V) interacts with both of them. Finally, we show for the first time that an AtBrca2 protein could bind two different partners at the same time: AtRad51 and AtDss1(I), or AtDmc1 and AtDss1(I).

Role of the N-terminal Domain of the Human DMC1 Protein in Octamer Formation and DNA Binding

The DMC1 protein, a eukaryotic homologue of RecA that shares significant amino acid identity with RAD51, exhibits two oligomeric DNA binding forms, an octameric ring and a helical filament. In the crystal structure of the octameric ring form, the DMC1 N-terminal domain (1-81 amino acid residues) was highly flexible, with multiple conformations. On the other hand, the N-terminal domain of Rad51 makes specific interactions with the neighboring ATPase domain in the helical filament structure. To gain insights into the functional role of the N-terminal domain of DMC1, we prepared a deletion mutant, DMC1-(82-340), that lacks the N-terminal 81 amino acid residues from the human DMC1 protein. Analytical ultracentrifugation experiments revealed that, whereas full-length DMC1 forms a octamer, DMC1-(82-340) is a heptamer. Furthermore, DNA binding experiments showed that DMC1-(82-340) was completely defective in both single-stranded and double-stranded DNA binding activities. Therefore, the N-terminal domain of DMC1 is required for the formation of the octamer, which may support the proper DNA binding activity of the DMC1 protein.

Activation of Human Meiosis-specific Recombinase Dmc1 by Ca2+

Rad51 and its meiotic homolog Dmc1 are key proteins of homologous recombination in eukaryotes. These proteins form nucleoprotein complexes on single-stranded DNA that promote a search for homology and that perform DNA strand exchange, the two essential steps of genetic recombination. Previously, we demonstrated that Ca2+ greatly stimulates the DNA strand exchange activity of human (h) Rad51 protein (Bugreev, D. V., and Mazin, A. V. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 9988-9993). Here, we show that the DNA strand exchange activity of hDmc1 protein is also stimulated by Ca2+. However, the mechanism of stimulation of hDmc1 protein appears to be different from that of hRad51 protein. In the case of hRad51 protein, Ca2+ acts primarily by inhibiting its ATPase activity, thereby preventing self-conversion into an inactive ADP-bound complex. In contrast, we demonstrate that hDmc1 protein does not self-convert into a stable ADP-bound complex. The results indicate that activation of hDmc1 is mediated through conformational changes induced by free Ca2+ ion binding to a protein site that is distinct from the Mg2+.ATP-binding center. These conformational changes are manifested by formation of more stable filamentous hDmc1.single-stranded DNA complexes. Our results demonstrate a universal role of Ca2+ in stimulation of mammalian DNA strand exchange proteins and reveal diversity in the mechanisms of this stimulation.