M I N I R E V I E W
From meiosis to postmeiotic events: Uncovering the molecular roles of the meiosis-specific recombinase Dmc1 Wataru Kagawa and Hitoshi Kurumizaka
Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo,Japan
Keywords helical filament; meiotic recombination; octameric ring; recombination regulators
Correspondence W. Kagawa and H. Kurumizaka, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan Fax: +81 3 5367 2820 Tel: +81 3 5369 7315 E-mail: wkagawa@aoni.waseda.jp; kurumizaka@waseda.jp
In meiosis, the accurate segregation of maternal and paternal chromosomes is accomplished by homologous recombination. A central player in meiotic recombination is the Dmc1 recombinase, a member of the RecA ⁄ Rad51 recombinase superfamily, which is widely conserved from viruses to humans. Dmc1 is a meiosis-specific protein that functions with the ubiqui- tously expressed homolog, the Rad51 recombinase, which is essential for both mitotic and meiotic recombination. Since its discovery, it has been speculated that Dmc1 is important for unique aspects of meiotic recombi- nation. Understanding the distinctive properties of Dmc1, namely, the fea- tures that distinguish it from Rad51, will further clarify the mechanisms of meiotic recombination. Recent structural, biochemical, and genetic findings are now revealing the molecular mechanisms of Dmc1-mediated homolo- gous recombination and its regulation by various recombination mediators.
(Received 30 July 2009, accepted 16 October 2009)
doi:10.1111/j.1742-4658.2009.07503.x
Introduction
In all sexually reproducing organisms, the number of chromosomes is halved in a specialized type of cell division called meiosis. This process of producing hap- loid cells ensures that each zygote has the same num- ber of chromosomes as its parents when the two haploid cells fuse during fertilization to form a diploid cell. Abnormalities in meiosis result in aneuploidy, a state with an abnormal number of homologous chro- mosomes. Many higher eukaryotes, including humans, have a low tolerance for aneuploidy, which generally results in developmental abnormalities and embryonic death at an early stage of development [1]. Thus, meio- sis is essential for genomic stability by maintaining the chromosome number through successive generations.
At the heart of meiosis is meiotic recombination. It establishes a physical connection between homologous chromosomes, which is the basis for their proper segre-
gation. The connections are established by a special- ized homologous recombination pathway (Fig. 1). Essentially, multiple DNA double-strand breaks (DSBs) are introduced throughout the chromosome by the activity of the Spo11 protein. Afterwards, one or more exonucleases process the DSBs to generate 3¢-ssDNA tails. DNA recombinases then bind to the extruded ssDNA tails, to form presynaptic filaments. The fila- ments bind to the intact double-stranded region of the homologous chromosome, and form new Watson–Crick base pairs (heteroduplex) between the ssDNA and the complementary strand of the dsDNA, in a step called homologous pairing. The heteroduplex region formed by homologous pairing is expanded by the succeeding step, referred to as DNA strand exchange. These two steps, homologous pairing and DNA strand exchange, are promoted by DNA recombinases, and are critical
Abbreviation DSB, double-strand break.
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Double-strand break
End resection
Homology search
D-loop formation
DNA synthesis
Holliday junction formation
Noncrossover
Crossover
Resolution
Fig. 1. DSB repair model for meiotic recom- bination. Meiotic recombination is initiated by the Spo11 protein, which cuts the DNA via a topoisomerase-like reaction to gener- ate a covalent protein–DNA linkage to the 5¢-DNA ends on either side of the break. Endonucleases then remove the Spo11 bound to short oligonucleotides. The DNA ends are processed by exonucleases, which yield 3¢-single-stranded tails. This region is bound by DNA recombinases (Dmc1 and Rad51), and the homologous region is searched for in the undamaged homologous chromosome (blue). Subsequent DNA synthesis and ligation yield the Holliday junction. Resolution of the Holliday junction yields either noncrossover or crossover products.
establishing the physical
connections between
for homologous chromosomes during meiosis.
characterized in vitro. RecA catalyzes
the synaptonemal complex [8]. It was speculated early on that Dmc1 is essential for these unique aspects of meiotic recombination. Several questions have arisen since the discovery of Dmc1. What part of the meiotic recombination reaction is catalyzed by Dmc1, but not by Rad51? Does Dmc1 have intrinsic biochemical properties that differentiate it from Rad51? What fac- tors modulate the activities of Dmc1, and how specific is the modulation with respect to Rad51?
The octameric ring form of Dmc1
homologous
for most
Most eukaryotes possess two recombinases, Rad51 and Dmc1. Homologs of these proteins are widely from viruses to humans. The conserved in nature, homologous pairing and DNA strand exchange activi- ties of the bacterial ortholog RecA have been exten- sively the homologous pairing reaction by first forming a right- handed helical filament on ssDNA, consisting of approximately six protomers per turn. Recent X-ray crystallographic studies of the RecA–ssDNA com- plexes revealed a globally stretched ssDNA structure that probably represents the structural intermediate responsible for homologous pairing with the dsDNA molecule [2]. The eukaryotic homolog, Rad51, is recombination required pathways in both mitotic and meiotic cells [3]. Thus far, in vitro studies of Rad51 have indicated that it is strikingly similar to RecA, both structurally and biochemically [4].
coordination of
Electron microscopic studies of human DMC1 yielded the first indications that Dmc1 and Rad51 have struc- tural differences (human proteins are in capital letters). DMC1 forms octameric rings in the absence of DNA, and stacked rings in the presence of DNA [9,10]. Atomic force microscopy also revealed similar struc- tures with yeast (Saccharomyces cerevisiae and Schizo- saccharomyces pombe) Dmc1 proteins, indicating that the ring-based structural organization of Dmc1 is con- to human [11,12]. By contrast, served from yeast RAD51 and its orthologs exist as rings consisting of six to eight protomers in the absence of DNA [13–15], but form helical filaments in the presence of DNA [14,16–20] (Table 1). No binding of Rad51 rings to DNA has been observed. Thus, the ring-based DMC1 structures formed on DNA are clearly different from those of the RAD51 orthologs, despite the high sequence identity (more than 50%) between DMC1 and RAD51. The octameric ring form of DMC1 was further characterized in detail by X-ray crystallography
By contrast, the other recombinase, Dmc1, is meio- sis-specific [5]. In mice, targeted mutation of the Dmc1 gene results in sterility, and the hallmarks of poorly repaired DSBs are apparent in reproductive cells [6,7]. Thus, Dmc1 is important for meiotic recombination. Meiotic recombination has several unique features that distinguish it from the homologous recombination that occurs in mitotically dividing cells. They include the strong preference for interhomolog recombination, and the recombination with meiosis- specific, higher-order chromosome structures, such as
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Table 1. Reported quaternary structures of the members of the RecA ⁄ Rad51 ⁄ Dmc1 recombinase family. NR, not reported.
)DNA
+DNA
Homo sapiens DMC1 S. cerevisiae Dmc1 Sc. pombe Dmc1 Su. solfataricus RadA M. voltae RadA Pyrococcus furiosus RadA H. sapiens RAD51 Escherichia coli RecA
Octameric ringa Octameric ring, helical filament Octameric ring Octameric ring, helical filamenta Helical filamenta Heptameric ringa Octameric ring Hexameric ring, helical filamenta
Stacked octameric rings, helical filament Stacked octameric rings, helical filament Stacked octameric rings, helical filament Stacked octameric rings, helical filament NR Helical filament Helical filament Helical filamenta
a Solved by X-ray crystallography.
Asn163
Arg192
Glu258
Tyr194
Fig. 2. The protomer–protomer interface of the DMC1 octameric ring. The two protomers (in the asymmetric unit of the crystal – Protein Data Bank ID: 1V5W) are colored orange and blue. The boxed area shows the location of the tripartite hydrogen-bonding network at the pro- tomer–protomer interface. The close-up view of the network is shown on the right. Glu258 hydrogen bonds with Asn163, Arg192 and Tyr194 from the neighboring protomer. Glu258 is conserved in yeast Dmc1 proteins and Su. solfataricus RadA, which are known to form octameric rings. All structural figures were created using the PYMOL program [70].
the protomer–protomer interface of
[21]. A tripartite hydrogen-bonding network was found at the DMC1 octameric ring (Fig. 2). The amino acid (Glu258) involved in this network is absent in RAD51, thus providing an explanation for the observation of stable octameric ring formation observed with DMC1, but not with RAD51. The electron microscopic and X-ray crystallographic studies of DMC1 both suggested a tail-to-tail (or head-to-head) association of the octa- meric rings on DNA [10,21], in which the DNA passes through the central channel of the stacked rings. How- ever, to date, no links have been made between these ring structures and the function of DMC1. Whether these structures are functionally relevant in DMC1- mediated homologous recombination remains to be elucidated.
whereas DMC1 exhibits significantly weaker strand exchange activity under these conditions [9]. The con- ditions required by DMC1 for efficient catalysis of the DNA strand exchange reaction were recently discov- ered. They include the presence of KCl at near-physio- logical ionic strength [22], or the inclusion of calcium ions [23]. Under these conditions, Dmc1 forms helical filaments on ssDNA that closely resemble those of the Rad51–ssDNA complex, as observed by electron microscopic studies [22,23]. Yeast (S. cerevisiae) Dmc1 also efficiently formed helical filaments on ssDNA in the presence of calcium ions [24]. These findings strongly suggest that Dmc1 promotes DNA strand exchange as helical filaments. These breakthroughs in the biochemical study of Dmc1 represent an important landmark, as they provided a foundation for further detailed characterizations of the recombinase.
The helical filament form of Dmc1
Another important difference between Dmc1 and Rad51 or RecA was revealed by in vitro DNA strand exchange assays. RecA displays optimal DNA strand exchange activity under relatively low-salt conditions,
The mechanism underlying the salt stimulation in Dmc1-mediated strand exchange is not fully under- stood. RAD51 also exhibits a salt (ammonium sulfate) requirement for enhanced DNA strand exchange activ- ity [25]. Subsequent studies revealed that this salt (ammonium sulfate) enhances the ability of RAD51 to
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distinguish ssDNA from dsDNA, and induces a conformational change in RAD51, leading to the formation of extended nucleoprotein filaments on ssDNA [26]. These observations were further sup- ported by studies on the effects of several neutral salts on RAD51 activities [27]. In the case of DMC1, a Ca2+ may bind to a specific site distinct from the ATP-binding site, and induce conformational changes in the protein that enhance its ability to form filamen- tous structures on ssDNA and efficiently promote DNA strand exchange [23]. This mechanism differs from the Ca2+ stimulation mechanism proposed for the RAD51 protein, in which the Ca2+ binds to the ATP-binding site and prevents the rapid conversion of the RAD51– ATP–ssDNA complex into the inactive ADP-bound form, thereby maintaining the active form [28].
Luo et al. have provided a structural basis for the K+ and Ca2+ stimulation of the DNA strand exchange promoted by a Dmc1 ortholog, Methanococ- cus voltae RadA. The crystal structure of RadA com- plexed with AMP–PNP, Mg2+ and K+ revealed that two K+ are bound to the c-phosphate of the non- hydrolyzable ATP analog, away from the Mg2+-bind- ing site [29,30]. The L2 loop, which directly binds to DNA and promotes DNA strand exchange, is quite disordered in the structure without potassium ions, but becomes ordered in the presence of potassium ions. The L2 loop also became ordered in the presence of Ca2+, which took the place of the two K+ [31].
ments in the presence of a nonhydrolyzable ATP ana- log, AMP-PNP, and calcium ions, in the absence of DNA [35]. Similar left-handed filaments were observed with the DMC1 ortholog, Sulfolobus solfataricus RadA. High-resolution structures of a left-handed heli- cal filament, along with a noncanonical, extended right-handed filament of RadA, were recently solved by X-ray crystallography [35]. The crystal structures revealed that the DNA-binding motifs (L1, L2, and helix-hairpin-helix) are all located on the exterior of their respective helical filaments. The exterior positions of the DNA-binding motifs agree well with the pro- posed facilitated DNA rotation model, in which RecA family proteins catalyze recombination reactions as motor proteins by promoting coordinated rotation between dsDNA and ssDNA. This model requires the presence of DNA-binding sites on the exterior of the filament [36]. Although it is still possible that novel binding sites exist on the exterior of the filament, the hypothesis that helical filaments undergo quaternary structural changes to relocate the known DNA-binding motifs during recombination appears to be a valid working model. Whether these Dmc1 helical filaments are functionally relevant in DMC1-promoted recombi- nation reactions remains to be verified. If so, then the question of whether differences exist between the non- canonical filaments of DMC1 and RAD51 becomes important. The high-resolution X-ray crystal structures of these helical filaments bound to DNA will be help- ful for assessment of their functional relevance.
Studies of DMC1 polymorphic mutants
indicating that
The studies presented above indicate that salts and ions, such as K+ and Ca2+, are important cofactors of DMC1 that regulate its ATP hydrolysis and DNA strand exchange activities. The in vitro DNA strand exchange conditions established for DMC1 have been tested with the plant Dmc1 proteins from rice, Oryza sativa [32–34]. These studies demonstrated that rice Dmc1 also requires K+ to efficiently promote DNA strand exchange by the formation of helical fila- ments [34], the ion requirement of Dmc1 is widely conserved.
Variations in the Dmc1 helical filaments
that
In humans, genome-wide single-nucleotide polymor- phism analyses have identified several DMC1 gene variants. Studying the effects of the mutations on the DMC1 activity may reveal important functional simi- larities and differences between RAD51 and DMC1. Recently, two variants, DMC1-M200V and DMC1- I37N (Fig. 3A,B), have been biochemically character- ized [37,38]. DMC1-M200V was identified in an infertile patient [39], and the possibility of the DMC1 infertility was recombinase activity as a source of examined. DMC1-M200V requires a higher Mg2+ con- centration for optimal D-loop formation activity. The crystal structure of the M. voltae RadA revealed a sec- ond Mg2+-binding site (apart from the ATP-binding site) is probably well conserved within the RecA ⁄ Rad51 ⁄ Dmc1 recombinase superfamily, and is essential for active helical filament formation [40]. The amino acids constituting this binding site are some- what conserved between RadA and Dmc1 ⁄ Rad51. The crystal structure of DMC1-M200V suggested that the
As stated above, Dmc1 forms toroidal rings and heli- cal filaments. It is generally accepted that members of the RecA ⁄ Rad51 ⁄ Dmc1 recombinase superfamily pro- mote recombination reactions as right-handed helical filaments, with approximately six protomers per helical turn. However, recent studies utilizing atomic force microscopy have suggested that structural variations of Dmc1 helical filaments can occur. S. cerevisiae Dmc1 forms both right-handed and left-handed helical fila-
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A
B
C
Arg181
Asp246 (Glu258)
90˚
Thr283 Asn242
His237
Met189 (Met200)
Fig. 3. The corresponding locations of the human DMC1 Met200 and Ile37 residues on the active M. voltae RadA helical filament. One heli- cal turn (composed of six molecules) of the M. voltae RadA helical filament structure (Protein Data Bank ID: 1XU4) is viewed from the side (A) and the top (B). The N-terminal domain (pink) harbors Ile37 (red). Ile37 faces outwards, away from the helical filament axis. By contrast, Met200 is located on the ATPase domain (cyan), close to the helical filament axis. (C) The possible allosteric effect of the DMC1 M200V mutation on the putative Mg2+-binding site. An Mg2+-binding site essential for ATPase activity was found at the protomer–protomer interface in M. voltae RadA. Several residues, including Asn242 and Asp246, at the protomer–protomer interface coordinate the Mg2+ (green sphere). These residues are located on the same a-helix that is anchored to the ATPase domain, via hydrophobic interactions between His237 and Met189. The DMC1 Met200 and Glu258 residues correspond to Met189 and Asp246 of M. voltae RadA, respectively. Notably, the DMC1 Glu258 is involved in the tripartite hydrogen-bonding network in the octameric ring form. Thus, Glu258 may have dual roles, functioning in both ring stabilization and in ATPase activity.
guiding Dmc1 and Rad51 to their distinct roles in vivo. Bishop et al. have recently utilized transmission electron microscopy to visualize and compare the Dmc1 fila- ments with those of Rad51 and RecA [41]. The Dmc1 filaments observed by transmission electron microscopy were fundamentally identical to those of Rad51 and RecA, containing approximately six protomers per turn and exhibiting similar structural parameters, including filament stiffness (persistence length), helical pitch, fila- ment diameter, DNA base pairs per helical turn, and helical handedness. This study supports the view that the functional differences between Dmc1 and Rad51 are more likely to result from the influence of distinct sets of regulatory proteins, rather than from the intrinsic differences in the filament structures. This conclusion is consistent with the fact that the DNA strand exchange reactions promoted by Dmc1 and Rad51 in vitro are strikingly similar.
M200V mutation has an allosteric effect on the putative, second Mg2+-binding site. Thus, destabilized Mg2+ binding may be the basis for the requirement for higher Mg2+ concentrations in the homologous pairing reaction promoted by DMC1-M200V [37] (Fig. 3C). The other DMC1 variant, DMC1-I37N, dis- plays different Ca2+ requirements from the wild-type protein in the D-loop formation reaction, and hydro- lyzes ATP even in the absence of DNA. As filament formation is critical to ATP hydrolysis, this finding suggests that DMC1-I37N is capable of forming active helical filaments even in the absence of DNA [38]. Ile37 is located in the distinct N-terminal domain of DMC1 (Fig. 3A). Thus, the N-terminal domain of DMC1 could play essential roles in regulating its recombination activities. The biological consequences of the M200V and I37N mutations could be quite informative. Furthermore, studies of other DMC1 polymorphic variants may reveal unexpected properties of DMC1, which may clarify the unique roles of DMC1 in meiotic recombination.
Regulators of Dmc1
In contrast to the idea that Dmc1 has intrinsic structural and biochemical differences from Rad51, regulatory proteins have been suggested to play a larger role in
Studies of Rad51 and its regulators have revealed that the regulators in vivo and in vitro profoundly affect its recombinase activity [42]. Recently, many Dmc1- interacting proteins have been identified and character- ized genetically and biochemically. Understanding how these factors affect the activity of Dmc1, and determin- ing the specificity of their effects with respect to Rad51, will be essential for defining the unique roles played by Dmc1 in meiotic recombination.
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Hop2 and Mnd1
possesses branch migration activity, which suggests the involvement of Rad54 in the late stages of homologous recombination [63]. RAD54B physically interacts with DMC1 in vitro, and stimulates its DNA strand exchange activity [64]. However, the functional signifi- cance of the RAD54B–DMC1 interaction remains to be determined, partly because there is little or no mei- otic recombination defect in mice lacking RAD54B [65].
BRCA2
Hop2 and Mnd1 were initially identified as chromo- some-associated factors [43]. The absence of these pro- teins causes nonhomologous chromosome synapsis and the persistence of meiotic DSBs [43–49]. Hop2 and Mnd1 form a heterodimeric complex, and the purified complex stimulates the D-loop formation and DNA strand exchange promoted by Dmc1 [44,50–55]. These observations suggest that Hop2 and Mnd1 function in the recombination pathway that is dependent on Dmc1. However, the molecular functions of Hop2 and Mnd1 still remain elusive. Currently, there are two working models: (a) the Hop2–Mnd1 complex has a presynaptic role in helping to load the Dmc1 recom- binase onto ssDNA; and (b) the Hop2–Mnd1 complex associates with the chromosome, and indirectly pro- motes the recombination activity of Dmc1.
Mei5 and Sae3
BRCA2 is a tumor suppressor protein, and several mutations of this protein are associated with a predis- position to breast and ovarian cancers. It consists of multiple BRC motifs (each composed of approximately 70 amino acids), which interact with RAD51. The crystal structure of RAD51 fused with one of the BRC motifs revealed that the motif interacts with RAD51 by mimicking the polymerization motif of RAD51 [66]. BRCA2 also has multiple copies of the OB fold, which is known to be an ssDNA-binding motif [67]. BRCA2 has been postulated to recruit RAD51 to DSB sites. Recently, a specific DMC1-interaction region in BRCA2 was identified [68].
Concluding remarks
Mei5 and Sae3 have been found in budding yeast; mei5 and sae3 mutants accumulate unrepaired meiotic DSBs, and lack the ability to form Dmc1 foci [56,57]. In contrast, Rad51 foci are still observed in both mutants, indicating that the roles of Mei5 and Sae3 are specific for the Dmc1-dependent pathway. Recently, the Mei5–Sae3 complex was reconstituted with the purified, recombinant proteins, and was shown to help load Dmc1 onto RPA-coated ssDNA [58].
Swi5 and Sfr1
that
the helical filaments
complex
The importance of Mei5 and Sae3 is underscored by the presence of its homologs in Sc. pombe. Two Mei5 homologs, Swi2 and Sfr1, and one Sae3 homolog, Swi5, have been identified. The purified recombinant the DNA strand stimulates Swi5–Sfr1 exchange mediated by both Rhp51 (Sc. pombe homo- log of Rad51) and Dmc1 [59]. These studies are consis- tent with the view that Swi5 and Sfr1, and their budding yeast homologs, have presynaptic roles in Rhp51-mediated or Dmc1-mediated recombination reactions.
Rad54 and its homologs
We are now at the stage where we can begin to deci- pher the structural and functional differences between Dmc1 and Rad51 in meiotic recombination. The important issue of whether the Dmc1 and Rad51 recombinases have intrinsic structural and biochemical differences has not been fully resolved. An emerging formed by the view is RecA ⁄ Rad51 ⁄ Dmc1 recombinase superfamily members are much more flexible than previously thought, form- ing right-handed and left-handed filaments containing various numbers of protomers per helical turn [69]. The differences could be revealed by the crystal struc- tures of the Dmc1 and Rad51 complexes with DNA, which recapitulate their respective in vivo states. On the other hand, another perspective suggests that the different meiotic functions of Dmc1 and Rad51 result from distinct sets of regulators acting on them, rather than their intrinsic differences as recombinases. To ver- ify this, we need a deeper mechanistic understanding of how the recombination regulators act on Dmc1 and Rad51. Structural information on the detailed inter- actions between Dmc1 or Rad51 and their regulators is certainly important. A large gap still exists between the meiotic phenomena observed in genetic studies the Dmc1 and the detailed molecular
studies of
Rad54, Rad54B and Rdh54 (also known as Tid1) are members of the SWI2 ⁄ SNF2 family of proteins, and are DNA-dependent ATPases. Rad54 stimulates the and DNA strand exchange homologous pairing promoted by Rad51 [60–62]. Furthermore, Rad54
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of the yeast Dmc1 protein ring and Dmc1–ssDNA nucleoprotein complex. Biochemistry 44, 6052–6058. 12 Sauvageau S, Stasiak AZ, Banville I, Ploquin M,
Stasiak A & Masson JY (2005) Fission yeast rad51 and dmc1, two efficient DNA recombinases forming helical nucleoprotein filaments. Mol Cell Biol 25, 4377–4387. 13 Shin DS, Pellegrini L, Daniels DS, Yelent B, Craig L,
recombinase. To bridge this gap, more sophisticated in vitro recombination reactions containing both Dmc1 and Rad51 along with their regulators will be impor- tant to clarify the complex interplay between these fac- tors. Furthermore, in vitro reactions that recapitulate the chromosome state during meiotic recombination should advance our understanding of the interactions between Dmc1, its regulators, and the structural pro- teins of the chromosome. Integrating the biochemical and structural findings with the functions revealed by genetics will illuminate the precise roles of Dmc1 in meiotic recombination.
Bates D, Yu DS, Shivji MK, Hitomi C, Arvai AS et al. (2003) Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2. EMBO J 22, 4566–4576.
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