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Fission yeast Swi5 protein, a novel DNA recombination mediator
d n a r e p a i r 7 ( 2 0 0 8 ) 1–9
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Mini-review
Fission yeast Swi5 protein, a novel DNA recombination mediator Nami Haruta a,1 , Yufuko Akamatsu a,2 , Yasuhiro Tsutsui b , Yumiko Kurokawa a , Yasuto Murayama a , Benoit Arcangioli c , Hiroshi Iwasaki a,∗ a b c
International Graduate School of Arts and Sciences, Yokohama City University, Tsurumi-ku, Yokohama 230-0045, Japan National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan Institut Pasteur, Paris Cedex 15, France
a r t i c l e
i n f o
a b s t r a c t
Article history:
The Schizosaccharomyces pombe Swi5 protein forms two distinct protein complexes, Swi5–Sfr1
Received 9 July 2007
and Swi5–Swi2, each of which plays an important role in the related but functionally dis-
Accepted 9 July 2007
tinct processes of homologous recombination and mating-type switching, respectively. The
Published on line 22 August 2007
Swi5–Sfr1 mediator complex has been shown to associate with the two RecA-like recombi-
Keywords:
mediated by these proteins. Genetic analysis indicates that Swi5–Sfr1 works independently
Swi5–Sfr1
of another mediator complex, Rhp55–Rhp57, during Rhp51-dependent recombinational
Rad51
repair. In addition, mutations affecting the two mediators generate distinct repair spectra of
Dmc1
HO endonuclease-induced DNA double strand breaks, suggesting that these recombination
Homologous recombination
mediators differently regulate recombination outcomes in an independent manner.
nases, Rhp51 (spRad51) and Dmc1, and to stimulate in vitro DNA strand exchange reactions
Recombination mediator
© 2007 Elsevier B.V. All rights reserved.
Schizosaccharomyces pombe
Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.
∗
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mating type switching and the S. pombe swi5 gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Two Swi5-interacting proteins in S. pombe. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Two sub-pathways dependent on Swi5–Sfr1 and Rhp55–Rhp57 in Rhp51-mediated recombinational repair in S. pombe . Swi5–Sfr1 and Rhp55–Rhp57 function redundantly in Rhp51 assembly on DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Purification of the Swi5–Sfr1 complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Swi5–Sfr1 complex stimulates Rhp51-dependent strand exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The mechanism of Rhp51-dependent strand exchange stimulated by the Swi5–Sfr1 complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . Function of the Swi5–Sfr1 complex in meiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 2 2 4 5 5 5 5 6
Corresponding author. Tel.: +81 45 508 7238; fax: +81 45 508 7369. E-mail address: [email protected] (H. Iwasaki). 1 Present address: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan. 2 Present address: Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA. 1568-7864/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2007.07.004
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10. The Swi5–Sfr1 complex also stimulates Dmc1-dependent strand exchange in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Functional differences among Rad22, Rhp55–Rhp57 and Swi5–Sfr1 mediators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.
Introduction
Various types of DNA rearrangements are vitally important biological processes. Homologous recombination (HR), a representative example, plays critical roles in the mitotic and meiotic cell cycles of all organisms. In meiosis, HR is essential for correct chromosome disjunction by establishing a physical connection between homologues at the first meiotic division. In addition, the high frequency of meiotic recombination contributes to intra-species diversity. On the other hand, the primary function of HR in mitotic cells is to repair DNA damage such as double-strand breaks (DSBs) or single strand gaps that could be generated by DNA damaging treatments or that could arise as a result of replication fork collapse. Mitotic HR is also required for programmed DNA rearrangements such as mating-type switching (MT switching) in yeast. HR-generated DNA rearrangements involve recombinases, essential proteins that carry out homology searches and mediate DNA strand exchange. The bacterial RecA protein, a well-known recombinase, shares structural similarity with the two principal eukaryotic recombinases, Rad51 and Dmc1. Rad51 functions in both mitotic and meiotic HR, while Dmc1 is a meiosis-specific recombinase. RecA-like recombinases form nucleoprotein filaments on regions of single-stranded DNA (ssDNA), often referred to as presynaptic filaments, which are the core structures that are involved in homology searches and strand exchange. However, once recombinogenic ssDNA regions are formed, they are immediately coated by an ssDNAbinding protein, Replication protein A (RPA). RPA has a higher affinity for ssDNA than does Rad51, and Rad51 cannot form a presynaptic filament with an ssDNA region that is already coated by RPA. Therefore, RPA acts as a negative factor in this context although it has also been shown to not only function positively but also to be an essential factor in HR. Several Rad51 accessory proteins, referred to as recombinase mediators, have been identified; these mediators overcome the inhibitory effect of RPA with respect to presynaptic filament formation by Rad51. The Rad52 protein and Rad55–Rad57 heterodimer in the yeast Saccharomyces cerevisiae and BRCA2 in higher eukaryotes are such accessory proteins for which mediator functions have been demonstrated (reviewed in [1,2]. Recently, two Swi5-containing protein complexes were found to be new accessory factors for recombinases in the fission yeast Schizosaccharomyces pombe. In this mini-review, we focus on the properties of the Swi5-containing mediators (Fig. 1).
2. Mating type switching and the S. pombe swi5 gene Cells of the S. pombe homothallic strains (designated h90 ) efficiently switch between P (plus) and M (minus) mating types
6 6 7 7 8
(MTs) [3,4]. MT switching achieved during the mitotic cell cycle by a highly regulated gene conversion event involving the transcriptionally active mat1 locus that uses information from one of two silent cassettes (mat2-P and mat3-M). Switching is initiated by a transient DNA break (probably DSB) associated with DNA replication and is assumed to involve mechanism similar to those that act in HR (reviewed in [5,6]). Previous genetic studies defined 11 mutants with a reduced rate of MT switching (swi mutants), which were assigned to three phenotypic classes (Ia, Ib and II) [7–9]. Mutations with Class Ia gene (swi1, -3 and -7) cause a reduced steady-state level of the switch-specific break at mat1 and these mutations are epistatic with Class Ib mutations, suggesting that the functions of these genes are required for proper DSB formation for an initiation of MT switching. Class Ib mutants (swi2, -5 and -6) have a normal rate of breaks formation at mat1 but switch less frequently. It was proposed that their products are required for efficient and proper utilization of the breaks in the initial steps of strand invasion that leads to gene conversion for MT switching. Class II (swi4, -8, -9 and -10 and rad22) mutants also have a normal rate of breaks, but produce a high proportion of heterothallic progeny containing extensive DNA rearrangements of the mating-type loci. These phenotypes imply that mutants in this class are error-prone in resolving of gene conversion intermediates formed during MT switching. The functions of these swi gene products revealed so far are summarized in Table 1. S. pombe Rad22 is an orthologue of S. cerevisiae Rad52 and both proteins play a critical role in HR. On the other hand, nine swi mutants exhibit normal HR, at least to outward appearance, indicating that they are not directly involved in HR, and only the swi5 mutant shows defects in HR and HRdependent repair as well as in MT switching [10]. Therefore, the Swi5 protein is a key matchmaker that creates a mechanistic link between HR and MT switching, both of which are DNA rearrangements mediated by Rhp51, the S. pombe Rad51 orthologue [11,12].
3. Two Swi5-interacting proteins in S. pombe The S. pombe swi5 gene encodes a small protein consisting of only 85 amino acids that is evolutionarily conserved from S. cerevisiae to humans [13,14]. The C-terminal half of Swi5 is particularly well conserved, and therefore this region may contain a novel motif with unknown functions. (Swi5 in S. pombe is homologous to S. cerevisiae Sae3 and unrelated to S. cerevisiae Swi5, a zinc finger transcription factor [15].) Swi5 physically interacts with the Swi2 protein, a 722 amino acid protein with a molecular size of 82 kDa [13]. Swi2 in S. pombe is homologous to S. cerevisiae Mei5 and unrelated to S. cerevisiae Swi2, a component of a SWI/SNF chromatin remod-
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Fig. 1 – Functions of Swi5-containing protein complexes. Each of the two complexes, Swi5–Sfr1 and Swi5–Swi2, interacts directly with the recombinase in an Sfr1- and Swi2-dependent manner, respectively, and acts as a recombinase mediator. The Swi5–Sfr1 mediator functions specifically for homologous recombination (HR) and HR repair while the Swi5–Swi2 complex functions specifically for mating type (MT) switching. The Rhp55–Rph57 heterodimer is also known as a mediator. One of the major mechanisms for DNA double strand breaks (DSB) is an HR-dependent double strand break repair (DSBR) during mitotic cell cycle. During mitotic HR repair, two mediators (Rhp55–Rhp57 or Swi5–Sfr1) promote independently the formation of active Rhp51 presynaptic filaments, leading to the formation of a D-loop, the initial DNA intermediate. If the initial D-loop is processed via synthesis-dependent single strand annealing mechanism (SDSA), recombinants without crossover (non-CO) are produced. SDSA is a major pathway in mitotic DSB repair. However, if the D-loop is converted into a further DNA intermediate via a so-called double strand break repair mechanisms (DSBR), a double Holiday structure is formed. A hypothesis is that this further processing of the D-loop may involve Rhp55–Rhp57, but not Swi5–Sfr1. This hypothesis agrees with an observation that rhp51 and rhp57 single mutants and rhp57 swi5 double mutant do not produce detectable crossover recombinants (CO) while swi5 and sfr1 single mutants still produce CO recombinants with a slightly reduced rate [19]. During meiosis, Rhp51 activated by Rhp55–Rhp57 and Dmc1 activated by Swi5–Sfr1 collaborate to produce CO recombinants. This is a major pathway in meiosis. In a subpathway, Rhp51 filaments may also be activated by Swi5–Sfr1. The activated Rhp51 filaments may be able to produce non-CO recombinant via SDSA. The details are sketchy. In mating type (MT) switching, the initial unstable Rhp51 filament is stabilized when it encounters the Swi5-Swi2 mediator that localizes on donor loci to form a D-loop like intermediate. This intermediate is processed via an SDSA-like pathway to produce non-CO type recombinant.
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Table 1 – S. pombe swi genes Class
Gene
Products and/or budding yeast (human) homologues
References
Class Ia
swi1+ swi3+ swi7+ sap1+
Tof1 (Timeless) Csm3 (Tipin) DNA polymerase ␣ Not found in budding yeast
[46,47] [48,49] [50,51] [52]
Class Ib
swi2+ swi5+ swi6+
Not found Sae3 Chromodomain protein (HP1). Not found in budding yeast
[13] [13,14] [17]
Class II
swi4+ swi8+ swi9+ (rad16+ ) swi10+ rad22+
Msh3 Msh2 Rad1 in budding yeast. ERCC4 (XPF) in vertebrates Rad10 in budding yeast. ERCC1 in vertebrates Rad52
[53] [54] [55] [56] [57]
eling factor [16]. The S. pombe Swi2 protein binds to Rhp51 and Swi6, in addition to Swi5. Swi6 is a homologue of heterochromatin protein 1 (HP1), found in higher multi-cellular eukaryotes [17]. The middle region of Swi2 is responsible for binding to Swi6 and the C-terminal region is involved in binding to Rhp51 and Swi5 [13]. Swi2 has two AT hook motifs in the N-terminal one-third region, which is assumed to be involved in DNA binding. All of these Swi proteins (Swi2, -5 and -6) belong to Class Ib group and have been suggested to play roles in processing DSB at the mat1 locus and the initiation steps of strand invasion with a correct donor selection (such that mat1P is converted to mat1-M and vice versa) through MT switching [6,18]. Swi2 and Swi6 form overlapping foci on nuclear heterochromatic regions that are also merged with Swi5 foci [19]. Chromatin immunoprecipitation analysis has revealed that the Swi2 and Swi5 proteins exhibits cell type-specific (P or M) localization patterns at the silent donor cassette region [18]. In P cells, Swi2/5 localization is restricted to a small region located adjacent to mat3-M, but in M cells, Swi2/5 is distributed across the entire silent mating-type interval that includes mat2-P in a heterochromatin-dependent manner. This differential localization of Swi2/5 with respect to regulatory interactions with Swi6 is suggested to be critical in modulating donor choice during MT switching [18]. Swi5 also interact with Sfr1 (Swi five-dependent recombination repair protein 1)[13]. Bacterially co-expressed Swi5 and Sfr1 were recently purified as a stable protein complex with a 2:1 stoichiometry [20]. Sfr1 consists of 299 amino acids, approximately half the size of Swi2. Interestingly, it shares similarity with the C-terminal half of Swi2, which overlaps the interaction region for Swi5 and Rhp51, but it does not contain the interaction region for Swi6 (Fig. 2). Indeed, Sfr1 directly interacts with Rhp51 but not Swi6 [13,20].
Fig. 2 – Schematic representations of Swi2 and Sfr1 proteins. The C-terminal half of Swi2 is homologous to Sfr1, which is the interaction site of Swi5 and Rhp51.
swi2 single mutants have a MT switching defect as described above, but have a normal repair activity in response to DNA damage such as that caused by exposure to ␥-ray or UV irradiation, or to methyl methanesulfonate (MMS). On the other hand, sfr1 single mutants undergo normal MT switching but are defective in DNA repair (see details below). Overproduction of Sfr1 cannot suppress the MT switching defect of the swi2 mutant, and overproduction of Swi2 cannot suppress DNA repair defect of the sfr1 mutant. These results indicate that the functions of Swi2 and Sfr1 are not redundant and they cannot crosstalk each other. The swi5 mutant is defective in both MT switching and DNA repair. Therefore, Swi5 functions like a switch button in two functions [13].
4. Two sub-pathways dependent on Swi5–Sfr1 and Rhp55–Rhp57 in Rhp51-mediated recombinational repair in S. pombe Both the first isolated S. pombe swi5-39 mutant with ochre mutation that is changed from Gln-38 and a swi5 deletion mutant newly constructed with using the cloned gene show very similar moderate sensitivity to DNA damaging agents such as UV irradiation, ␥-ray irradiation and methyl methanesulfonate (MMS), but its sensitivity is milder than that of the rhp51 single mutant [10,13]. The swi5 rhp51 double mutant is as sensitive as the rhp51 single mutant to these DNA damaging treatments. These epistatic relationships suggest that Swi5 functions in an Rhp51-dependent recombinational repair pathway [13]. Both the swi5 and sfr1 single mutants have very similar DNA repair defects each other, and the swi5 sfr1 double mutant has almost the same damage sensitivity as each single mutant. In addition, the swi5 rhp51 double mutant and the swi5 sfr1 rhp51 triple mutant have the same damage sensitivity as the rhp51 single mutant. These results provide rigorous genetic evidence that the Swi5–Sfr1 complex functions in the Rhp51-dependent recombination pathway [13]. Previous works have shown that the Rhp55–Rhp57 heterodimer is implicated in the Rhp51-dependent recombination pathway [21–23]. S. pombe Rhp55 and Rhp57 are counterparts of S. cerevisiae Rad55 and Rad57, respectively, and
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they all are referred to as Rad51 paralogues because of their amino acid sequence similarity [21,22,24]. Various genetic and cytological studies suggest that the Rad55–Rad57 heterodimer functions in the Rad51-dependent recombination pathway as an accessory factor for Rad51 (reviewed in [25]). P. Sung biochemically demonstrated that the S. cerevisiae Rad55–Rad57 heterodimer assists Rad51 nucleoprotein filament formation in vitro, suggesting to act as a recombination mediator [26]. Therefore, the Rhp55–Rhp57 heterodimer is also implicated in the similar mediator function. What is the relationship between the two Rhp51dependent recombination sub-pathways that involve Swi5–Sfr1 complex and the Rhp55–Rhp57 heterodimer? Genetic analysis clearly indicates their relationships during recombinational repair. Each of the rhp55, rhp57, swi5 and sfr1 single mutants shows modest sensitivity to DNA damaging agents such as UV, ␥-ray and MMS, with rhp55 and rhp57 slightly more sensitive than swi5 and sfr1. The swi5 rhp57 and sfr1 rhp57 double mutants and the swi5 rhp57 rhp51 triple mutant show the very similar sensitivity as the rhp51 single mutant. Overproduction of Rhp57 does not suppress the swi5 or sfr1 mutation, and overproduction of Swi5 and/or Sfr1 does not suppress the rhp57 mutation. Taken together, these results indicate that Rhp51-dependent recombination has two parallel subpathways, one that is Rhp55–Rhp57-dependent and another that is Swi5–Sfr1 dependent [13].
5. Swi5–Sfr1 and Rhp55–Rhp57 function redundantly in Rhp51 assembly on DNA A recent cytological study of formation of Rhp51 foci after DNA damage shows that Swi5–Sfr1 is involved in Rhp51 assembly at damaged sites [19]. Almost all wild-type cells exhibited significant Rhp51 focal signals in nuclei, with about half of the nuclei having very strong signals, at a relatively high dose of UV irradiation (200 J/m2 ). In the same experimental condition, the frequency of swi5 cells with intense nuclear Rhp51 foci was strongly reduced and the frequency of the cell with weak Rhp51 focal signals are increased; then, the total frequency of cells with Rhp51 signals was only reduced in swi5 cells. Cells with the rhp57 mutation also showed a reduction in Rhp51 assembly that was much milder than what is seen in swi5 cells. The swi5 rhp57 double mutant is more severely affected than either single mutant. In these cells, only a few nuclei with strong Rhp51 foci have been observed although they can partially form weak Rhp51 foci. These results suggest that Swi5 and Rhp57 facilitate or stabilize Rhp51 nucleoprotein filament formation in a redundant manner (Fig. 1) [13,19].
6.
Purification of the Swi5–Sfr1 complex
We found that production of bacterially expressed Sfr1 protein was very poor while recombinant Swi5 alone was efficiently expressed in E. coli. Even worse, the small amount of Sfr1 produced was found to be insoluble. However, co-expression of Swi5 and Sfr1 allowed for a simple purification, in which Sfr1
5
could be overexpressed as a stable complex with Swi5 and the complex was recovered in a soluble fraction. The two proteins were co-purified during all purification steps and the complex can keep an oligomeric form in a solution at very high salt concentrations such as in 1.5 M NaCl [20]. The purified Swi5–Sfr1 complex has ssDNA- and dsDNAbinding activity, although it has no apparent preference for ssDNA or dsDNA. Since Swi5 alone does not show DNA binding activity, Sfr1 is assumed to have a DNA binding domain(s) (unpublished data).
7. The Swi5–Sfr1 complex stimulates Rhp51-dependent strand exchange It has been reported that RPA stimulates DNA strand exchange with long (plasmid-sized) DNA substrates, which can be mediated by from budding yeast or human Rad51 ([27,28], reviewed in [29]). However, the effect of RPA is very subtle in the case of reactions mediated by S. pombe proteins: the intermediates and products of the strand exchange reaction mediated by Rhp51 are almost undetectable, whether carried out in the presence or absence of RPA. In turn, the addition of the Swi5–Sfr1 complex greatly enhances the DNA strand exchange reaction [20]. The optimal concentration is only about 1/20 to 1/10 the concentration of Rhp51 and excess concentrations of Swi5–Sfr1 inhibit the reaction. Note that this reaction still requires RPA. Generally, Rad51-mediated strand-exchange requires ATP, and the S. pombe Rhp51-mediated reaction in the presence of Swi5–Sfr1 is no exception. Since the Swi5–Sfr1 complex alone does not have DNA-strand exchange activity or ATPase activity, the Swi5–Sfr1 complex is suggested to stimulate the Rhp51-dependent strand exchange reaction, but not to promote strand exchange by itself. However, it remains to be determined whether the Swi5–Sfr1 complex is a bona fide mediator, as discussed below.
8. The mechanism of Rhp51-dependent strand exchange stimulated by the Swi5–Sfr1 complex Rad51 from budding yeast and from human cells has an ATPase activity that is greatly enhanced by ssDNA: in the absence of ssDNA and in the presence of dsDNA, their ATPase is very low [30,31]. However, S. pombe Rhp51 has a DNA-independent ATPase activity, which is only modestly enhanced by ssDNA or dsDNA [20,32]. The Swi5–Sfr1 complex enhances the Rhp51 ATPase activity in the presence of ssDNA by two-to three-fold, although it does not affect the activity in the presence of dsDNA or in the absence of DNA. Interestingly, the Swi5–Sfr1 complex does not quantitatively affect the ssDNA binding activity of Rhp51. The binding of Rhp51 is almost saturated at three nucleotides per Rad51 monomer in either the absence or presence of Swi5–Sfr1. Therefore, the Swi5–Sfr1complex does not simply increase the ssDNA binding by Rhp51, but rather it somehow alters the Rhp51 filament, which is referred to as “activation” in many biological reactions.
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Is the Swi5–Sfr1 complex, a canonical recombination mediator? The assessment of the mediator activity of Swi5–Sfr1 is, in very real sense, complicated since the Rhp51-mediated strand exchange reaction greatly depends on Swi5–Sfr1 under experimental conditions. When RPA is added later, which is experimentally considered to reduce its inhibitory effect, the reaction still requires Swi5–Sfr1. Therefore, it is difficult to evaluate the overall RPA inhibitory effect on Rhp51-mediated strand exchange reaction in the absence of Swi5–Sfr1. However, when RPA is added to ssDNA first, the product yield is greatly reduced. In this case, long incubation with Rhp51 and Swi5–Sfr1 before starting the reaction gradually restores strand exchange, while the addition of Swi5–Sfr1 after a long incubation of Rph51 does not. These properties indicate that Swi5–Sfr1 has a potential mediator activity [20].
9. Function of the Swi5–Sfr1 complex in meiosis S. cerevisiae Sae3 and Mei5 are Swi5 and Sfr1 homologues, respectively. Sae3 shares sequence homology with the whole region of Swi5 (Swi5 versus Sae3 24%/53% for identical/similar amino acids), while high sequence homology is shared only between the Sfr1 and Mei5 C-terminal regions (Sfr1 versus Mei5 24%/47% for identical/similar amino acids). In addition, overall sequence homologies are relatively low [13,33,34]. Sae3 and Mei5 are expressed only during meiosis and seems to be specific for meiotic recombination associated with a meiosis-specific recombinase Dmc1 [33,34]. The sae3 and mei5 mutants both show phenotypes very similar to that of the dmc1 mutant, which includes reductions in sporulation, spore viability and cross over recombination. Dmc1, Sae3 and Mei5 forms interdependently foci and colocalize on chromosomes in early steps of meiotic division. Taken together, Sae3 and Mei4 are implicated to form complex with Dmc1 and to stimulate Dmc1 assembly to the DSB sites [33,34]. By an analogy to the mitotic function of the S. cerevisiae homologue, S. pombe Swi5 is also expected to act as the mediator for the meiotic-specific recombinase, Dmc1. However, swi5 mutants exhibit severer defects in spore viability, meiotic recombination frequency and the DSB repair than dmc1 mutants, indicating that swi5 is epistatic to dmc1 [14,35]. Thus, Swi5 obviously has the additional function. The rhp55 swi5 and rhp57 swi5 double mutants drastically reduce their spore viability, implying a redundant function between Rhp55–Rhp57 heterodimer and Swi5 [14]. An idea that Swi5 is directly involved in presynaptic formations of both Rhp51 and Dmc1 is well in accordance with the above observation (Fig. 1). What is a meiotic partner with Swi5? A swi2 mutant has no reduction in meiotic recombination [36]. On the other hand, sfr1 has also been identified as mug13, a mutant exhibiting aberrant chromosome segregation in meiosis [37]. The sfr1 mutant reduces meiotic recombination frequency (our unpublished data). In addition, we have shown that the purified Swi5–Sfr1 complex interacts with Dmc1 in an Sfr1-dependent manner [20]. Thus, it is concluded that Sfr1 partners with Swi5 in meiosis as well.
10. The Swi5–Sfr1 complex also stimulates Dmc1-dependent strand exchange in vitro Indeed, the Swi5–Sfr1 complex was found to stimulate Dmc1mediated strand exchange in much the similar way that it affects the Rhp51-mediated strand exchange reaction [20]. In contrast to its effect on Rhp51, interestingly, it enhanced the ssDNA binding activity of Dmc1. Dmc1 has no, or very little, ATPase activity in the absence of DNA and its ATPase activity requires ssDNA that be bound by Dmc1. The ssDNAdependent ATPase activity of Dmc1 is inhibited by RPA, and Swi5–Sfr1 restores the activity, suggesting it overcomes the RPA inhibitory effect [20]. Although we did not directly demonstrate this in a previous paper, we have recently found that Swi5–Sfr1 stimulates Dmc1-mediated strand exchange using ssDNA pre-coated with RPA under certain experimental conditions (unpublished data). This finding indicates that Swi5–Sfr1 assists in loading Dmc1 onto ssDNA prebound with RPA. In other words, the Swi5–Sfr1 is a canonical mediator at least for Dmc1.
11. Functional differences among Rad22, Rhp55–Rhp57 and Swi5–Sfr1 mediators Another S. pombe mediator is Rad22, a homologue of Rad52 proteins in other eukaryotes including, budding yeast and humans. Genetic studies have indicated that Rad22 is involved in each of two parallel Rhp51-dependent sub-pathways during recombinational repair in S. pombe [38,39]. Therefore, a pair of two mediators (Rad22/Rhp55–Rhp57 and Rad22/Swi5–Sfr1) works in each sub-pathway. What are the functional differences among these mediators? Why are two mediators required in each pathway? A clue comes from our recent biochemical studies (unpublished data). A low efficiency of an Rhp51–Swi5–Sfr1-mediated strand exchange reaction, in which ssDNA is preincubated with RPA, is greatly recovered by the addition of Rad22. On the other hand, Rad52 from budding yeast and humans has been demonstrated to act as a Rad51 mediator, leading to a high yield of strand exchange products [40–43]. However, S. pombe Rad22 alone does not stimulate the Rhp51-mediated strand exchange. Swi5–Sfr1 still requires a full Rhp51-mediated strand exchange reaction. These results suggest that the molecular roles of the two mediators are different, and that both are essential for full Rhp51-mediated strand exchange activity (unpublished data). A working hypothesis is as follows: Rad22 is recruited onto RPA-coated ssDNA regions via a high affinity for RPA, allowing loading of inactive Rhp51. Subsequently, Swi5–Sfr1 activates the Rhp51, which promotes to extend and stabilize Rhp51 nucleofilament with concomitant disassembly of RPA from ssDNA. We assume that Rhp55–Rhp57 plays a biochemical role similar to that of Swi5–Sfr1 in Rhp51-dependent strand exchange. Further biochemical studies will be necessary to understand the molecular function of Rhp55–Rhp57 in the context of Rad22 mediator-dependent HR pathway. How do the in vivo functions of Rhp55–Rhp57 and Swi5–Sfr1 differ? We examined products generated by HO endonucleaseinduced DSBs in these mutant cells, an assay that was
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7
Fig. 3 – A model for a action of Swi5–Sfr1 on the two mediators. Rhp51 loads onto ssDNA by itself as an inactive filament and the Swi5–Sfr1 complex activates the Rhp51 filament. On the other hand, the Swi5–Sfr1 mediator directly assists Dmc1 to load onto ssDNA. The resultant Dmc1 filament that contains Swi5–Sfr1 is an active form.
originally developed by the Humphrey group [44]. Canonical crossover recombinational repair products could be detected at low but significant frequencies in wild-type, swi5 and sfr1 strains, while they were not detected in rhp57 and rhp51 single mutants or in the rhp57 swi5 double mutant, suggesting that Rhp51 and Rhp57, but not Swi5–Sfr1, are essential for crossover production. These results suggest that Swi5–Sfr1 processes DSBs in a manner different from that of the Rhp55–57 mediator (Fig. 1) [19]. However, at about the same time, the Freyer group independently reported that the rhp57 cells produce more cross over products than wild type cells in the same HO-induced DSB repair assay [45]. The reason for this discrepancy is unclear.
12.
Perspective
The apparent effects of the Swi5–Sfr1 accessory factor with respect to Rhp51 and Dmc1 are different. However, this may be only one point of view, and the fundamental contribution of Swi5–Sfr1 to the two recombinases could be the same: to promote the formation of an active nucleoprotein filament (Fig. 3). The differences between the two recombinases might be due to differences in their abilities to promote filament formation. Rhp51 alone has the potential to form a nucleoprotein filament, but Dmc1 does not. Dmc1 easily forms rings or stacked-rings in the presence of DNA and does not readily form helical filaments. Swi5–Sfr1 assists in the formation of the active helical filament from the Dmc1 stacked structure, and the helical filament structure of Dmc1 is the active form for strand exchange. On the other hand, because Rhp51 has the potential to form a nucleoprotein filament by itself,
and Swi5–Sfr1 may serve only to activate the inactive Rhp51 filament. A central question concerns why both recombinases are required in meiosis. The fundamental biochemical functions of Rad51 and Dmc1 are very similar and there are no significant differences between them, at least based our biochemical knowledge of each protein. Many accessory factors could control each recombinase to work cooperatively in a spatial and temporal manner. Swi5–Sfr1 is a strong candidate as a key to understanding the control mechanisms of these two recombinases in meiosis. Swi5 forms a protein complex with Swi2, which participates in MT switching, a gene conversion event not associated with crossover. It has been speculated that Swi5–Swi2 also functions as a mediator in the MT switching reaction, which also involves Rhp51. Determination of further biochemical functions of Swi2 will shed light on a role of Swi5 in influencing homologous recombination and mating type switching. In conclusion, Swi5 is a newly identified global regulator in DNA rearrangement reactions that involve RecA-type recombinases. Molecular analysis of DNA rearrangement mechanisms involving Swi5 has just started.
Acknowledgements Our study was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology (MECSST) of Japan and from the Japan Society for the Promotion of Science (JSPS), and by a grant for the 2005 Strategic Research Project (no. K17005) of Yokohama City University. NH was supported by JSPS fellowships for young scientists.
8
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available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/dnarepair
Mini-review
Fission yeast Swi5 protein, a novel DNA recombination mediator Nami Haruta a,1 , Yufuko Akamatsu a,2 , Yasuhiro Tsutsui b , Yumiko Kurokawa a , Yasuto Murayama a , Benoit Arcangioli c , Hiroshi Iwasaki a,∗ a b c
International Graduate School of Arts and Sciences, Yokohama City University, Tsurumi-ku, Yokohama 230-0045, Japan National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan Institut Pasteur, Paris Cedex 15, France
a r t i c l e
i n f o
a b s t r a c t
Article history:
The Schizosaccharomyces pombe Swi5 protein forms two distinct protein complexes, Swi5–Sfr1
Received 9 July 2007
and Swi5–Swi2, each of which plays an important role in the related but functionally dis-
Accepted 9 July 2007
tinct processes of homologous recombination and mating-type switching, respectively. The
Published on line 22 August 2007
Swi5–Sfr1 mediator complex has been shown to associate with the two RecA-like recombi-
Keywords:
mediated by these proteins. Genetic analysis indicates that Swi5–Sfr1 works independently
Swi5–Sfr1
of another mediator complex, Rhp55–Rhp57, during Rhp51-dependent recombinational
Rad51
repair. In addition, mutations affecting the two mediators generate distinct repair spectra of
Dmc1
HO endonuclease-induced DNA double strand breaks, suggesting that these recombination
Homologous recombination
mediators differently regulate recombination outcomes in an independent manner.
nases, Rhp51 (spRad51) and Dmc1, and to stimulate in vitro DNA strand exchange reactions
Recombination mediator
© 2007 Elsevier B.V. All rights reserved.
Schizosaccharomyces pombe
Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.
∗
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mating type switching and the S. pombe swi5 gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Two Swi5-interacting proteins in S. pombe. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Two sub-pathways dependent on Swi5–Sfr1 and Rhp55–Rhp57 in Rhp51-mediated recombinational repair in S. pombe . Swi5–Sfr1 and Rhp55–Rhp57 function redundantly in Rhp51 assembly on DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Purification of the Swi5–Sfr1 complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Swi5–Sfr1 complex stimulates Rhp51-dependent strand exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The mechanism of Rhp51-dependent strand exchange stimulated by the Swi5–Sfr1 complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . Function of the Swi5–Sfr1 complex in meiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 2 2 4 5 5 5 5 6
Corresponding author. Tel.: +81 45 508 7238; fax: +81 45 508 7369. E-mail address: [email protected] (H. Iwasaki). 1 Present address: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan. 2 Present address: Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA. 1568-7864/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2007.07.004
2
d n a r e p a i r 7 ( 2 0 0 8 ) 1–9
10. The Swi5–Sfr1 complex also stimulates Dmc1-dependent strand exchange in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Functional differences among Rad22, Rhp55–Rhp57 and Swi5–Sfr1 mediators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.
Introduction
Various types of DNA rearrangements are vitally important biological processes. Homologous recombination (HR), a representative example, plays critical roles in the mitotic and meiotic cell cycles of all organisms. In meiosis, HR is essential for correct chromosome disjunction by establishing a physical connection between homologues at the first meiotic division. In addition, the high frequency of meiotic recombination contributes to intra-species diversity. On the other hand, the primary function of HR in mitotic cells is to repair DNA damage such as double-strand breaks (DSBs) or single strand gaps that could be generated by DNA damaging treatments or that could arise as a result of replication fork collapse. Mitotic HR is also required for programmed DNA rearrangements such as mating-type switching (MT switching) in yeast. HR-generated DNA rearrangements involve recombinases, essential proteins that carry out homology searches and mediate DNA strand exchange. The bacterial RecA protein, a well-known recombinase, shares structural similarity with the two principal eukaryotic recombinases, Rad51 and Dmc1. Rad51 functions in both mitotic and meiotic HR, while Dmc1 is a meiosis-specific recombinase. RecA-like recombinases form nucleoprotein filaments on regions of single-stranded DNA (ssDNA), often referred to as presynaptic filaments, which are the core structures that are involved in homology searches and strand exchange. However, once recombinogenic ssDNA regions are formed, they are immediately coated by an ssDNAbinding protein, Replication protein A (RPA). RPA has a higher affinity for ssDNA than does Rad51, and Rad51 cannot form a presynaptic filament with an ssDNA region that is already coated by RPA. Therefore, RPA acts as a negative factor in this context although it has also been shown to not only function positively but also to be an essential factor in HR. Several Rad51 accessory proteins, referred to as recombinase mediators, have been identified; these mediators overcome the inhibitory effect of RPA with respect to presynaptic filament formation by Rad51. The Rad52 protein and Rad55–Rad57 heterodimer in the yeast Saccharomyces cerevisiae and BRCA2 in higher eukaryotes are such accessory proteins for which mediator functions have been demonstrated (reviewed in [1,2]. Recently, two Swi5-containing protein complexes were found to be new accessory factors for recombinases in the fission yeast Schizosaccharomyces pombe. In this mini-review, we focus on the properties of the Swi5-containing mediators (Fig. 1).
2. Mating type switching and the S. pombe swi5 gene Cells of the S. pombe homothallic strains (designated h90 ) efficiently switch between P (plus) and M (minus) mating types
6 6 7 7 8
(MTs) [3,4]. MT switching achieved during the mitotic cell cycle by a highly regulated gene conversion event involving the transcriptionally active mat1 locus that uses information from one of two silent cassettes (mat2-P and mat3-M). Switching is initiated by a transient DNA break (probably DSB) associated with DNA replication and is assumed to involve mechanism similar to those that act in HR (reviewed in [5,6]). Previous genetic studies defined 11 mutants with a reduced rate of MT switching (swi mutants), which were assigned to three phenotypic classes (Ia, Ib and II) [7–9]. Mutations with Class Ia gene (swi1, -3 and -7) cause a reduced steady-state level of the switch-specific break at mat1 and these mutations are epistatic with Class Ib mutations, suggesting that the functions of these genes are required for proper DSB formation for an initiation of MT switching. Class Ib mutants (swi2, -5 and -6) have a normal rate of breaks formation at mat1 but switch less frequently. It was proposed that their products are required for efficient and proper utilization of the breaks in the initial steps of strand invasion that leads to gene conversion for MT switching. Class II (swi4, -8, -9 and -10 and rad22) mutants also have a normal rate of breaks, but produce a high proportion of heterothallic progeny containing extensive DNA rearrangements of the mating-type loci. These phenotypes imply that mutants in this class are error-prone in resolving of gene conversion intermediates formed during MT switching. The functions of these swi gene products revealed so far are summarized in Table 1. S. pombe Rad22 is an orthologue of S. cerevisiae Rad52 and both proteins play a critical role in HR. On the other hand, nine swi mutants exhibit normal HR, at least to outward appearance, indicating that they are not directly involved in HR, and only the swi5 mutant shows defects in HR and HRdependent repair as well as in MT switching [10]. Therefore, the Swi5 protein is a key matchmaker that creates a mechanistic link between HR and MT switching, both of which are DNA rearrangements mediated by Rhp51, the S. pombe Rad51 orthologue [11,12].
3. Two Swi5-interacting proteins in S. pombe The S. pombe swi5 gene encodes a small protein consisting of only 85 amino acids that is evolutionarily conserved from S. cerevisiae to humans [13,14]. The C-terminal half of Swi5 is particularly well conserved, and therefore this region may contain a novel motif with unknown functions. (Swi5 in S. pombe is homologous to S. cerevisiae Sae3 and unrelated to S. cerevisiae Swi5, a zinc finger transcription factor [15].) Swi5 physically interacts with the Swi2 protein, a 722 amino acid protein with a molecular size of 82 kDa [13]. Swi2 in S. pombe is homologous to S. cerevisiae Mei5 and unrelated to S. cerevisiae Swi2, a component of a SWI/SNF chromatin remod-
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3
Fig. 1 – Functions of Swi5-containing protein complexes. Each of the two complexes, Swi5–Sfr1 and Swi5–Swi2, interacts directly with the recombinase in an Sfr1- and Swi2-dependent manner, respectively, and acts as a recombinase mediator. The Swi5–Sfr1 mediator functions specifically for homologous recombination (HR) and HR repair while the Swi5–Swi2 complex functions specifically for mating type (MT) switching. The Rhp55–Rph57 heterodimer is also known as a mediator. One of the major mechanisms for DNA double strand breaks (DSB) is an HR-dependent double strand break repair (DSBR) during mitotic cell cycle. During mitotic HR repair, two mediators (Rhp55–Rhp57 or Swi5–Sfr1) promote independently the formation of active Rhp51 presynaptic filaments, leading to the formation of a D-loop, the initial DNA intermediate. If the initial D-loop is processed via synthesis-dependent single strand annealing mechanism (SDSA), recombinants without crossover (non-CO) are produced. SDSA is a major pathway in mitotic DSB repair. However, if the D-loop is converted into a further DNA intermediate via a so-called double strand break repair mechanisms (DSBR), a double Holiday structure is formed. A hypothesis is that this further processing of the D-loop may involve Rhp55–Rhp57, but not Swi5–Sfr1. This hypothesis agrees with an observation that rhp51 and rhp57 single mutants and rhp57 swi5 double mutant do not produce detectable crossover recombinants (CO) while swi5 and sfr1 single mutants still produce CO recombinants with a slightly reduced rate [19]. During meiosis, Rhp51 activated by Rhp55–Rhp57 and Dmc1 activated by Swi5–Sfr1 collaborate to produce CO recombinants. This is a major pathway in meiosis. In a subpathway, Rhp51 filaments may also be activated by Swi5–Sfr1. The activated Rhp51 filaments may be able to produce non-CO recombinant via SDSA. The details are sketchy. In mating type (MT) switching, the initial unstable Rhp51 filament is stabilized when it encounters the Swi5-Swi2 mediator that localizes on donor loci to form a D-loop like intermediate. This intermediate is processed via an SDSA-like pathway to produce non-CO type recombinant.
4
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Table 1 – S. pombe swi genes Class
Gene
Products and/or budding yeast (human) homologues
References
Class Ia
swi1+ swi3+ swi7+ sap1+
Tof1 (Timeless) Csm3 (Tipin) DNA polymerase ␣ Not found in budding yeast
[46,47] [48,49] [50,51] [52]
Class Ib
swi2+ swi5+ swi6+
Not found Sae3 Chromodomain protein (HP1). Not found in budding yeast
[13] [13,14] [17]
Class II
swi4+ swi8+ swi9+ (rad16+ ) swi10+ rad22+
Msh3 Msh2 Rad1 in budding yeast. ERCC4 (XPF) in vertebrates Rad10 in budding yeast. ERCC1 in vertebrates Rad52
[53] [54] [55] [56] [57]
eling factor [16]. The S. pombe Swi2 protein binds to Rhp51 and Swi6, in addition to Swi5. Swi6 is a homologue of heterochromatin protein 1 (HP1), found in higher multi-cellular eukaryotes [17]. The middle region of Swi2 is responsible for binding to Swi6 and the C-terminal region is involved in binding to Rhp51 and Swi5 [13]. Swi2 has two AT hook motifs in the N-terminal one-third region, which is assumed to be involved in DNA binding. All of these Swi proteins (Swi2, -5 and -6) belong to Class Ib group and have been suggested to play roles in processing DSB at the mat1 locus and the initiation steps of strand invasion with a correct donor selection (such that mat1P is converted to mat1-M and vice versa) through MT switching [6,18]. Swi2 and Swi6 form overlapping foci on nuclear heterochromatic regions that are also merged with Swi5 foci [19]. Chromatin immunoprecipitation analysis has revealed that the Swi2 and Swi5 proteins exhibits cell type-specific (P or M) localization patterns at the silent donor cassette region [18]. In P cells, Swi2/5 localization is restricted to a small region located adjacent to mat3-M, but in M cells, Swi2/5 is distributed across the entire silent mating-type interval that includes mat2-P in a heterochromatin-dependent manner. This differential localization of Swi2/5 with respect to regulatory interactions with Swi6 is suggested to be critical in modulating donor choice during MT switching [18]. Swi5 also interact with Sfr1 (Swi five-dependent recombination repair protein 1)[13]. Bacterially co-expressed Swi5 and Sfr1 were recently purified as a stable protein complex with a 2:1 stoichiometry [20]. Sfr1 consists of 299 amino acids, approximately half the size of Swi2. Interestingly, it shares similarity with the C-terminal half of Swi2, which overlaps the interaction region for Swi5 and Rhp51, but it does not contain the interaction region for Swi6 (Fig. 2). Indeed, Sfr1 directly interacts with Rhp51 but not Swi6 [13,20].
Fig. 2 – Schematic representations of Swi2 and Sfr1 proteins. The C-terminal half of Swi2 is homologous to Sfr1, which is the interaction site of Swi5 and Rhp51.
swi2 single mutants have a MT switching defect as described above, but have a normal repair activity in response to DNA damage such as that caused by exposure to ␥-ray or UV irradiation, or to methyl methanesulfonate (MMS). On the other hand, sfr1 single mutants undergo normal MT switching but are defective in DNA repair (see details below). Overproduction of Sfr1 cannot suppress the MT switching defect of the swi2 mutant, and overproduction of Swi2 cannot suppress DNA repair defect of the sfr1 mutant. These results indicate that the functions of Swi2 and Sfr1 are not redundant and they cannot crosstalk each other. The swi5 mutant is defective in both MT switching and DNA repair. Therefore, Swi5 functions like a switch button in two functions [13].
4. Two sub-pathways dependent on Swi5–Sfr1 and Rhp55–Rhp57 in Rhp51-mediated recombinational repair in S. pombe Both the first isolated S. pombe swi5-39 mutant with ochre mutation that is changed from Gln-38 and a swi5 deletion mutant newly constructed with using the cloned gene show very similar moderate sensitivity to DNA damaging agents such as UV irradiation, ␥-ray irradiation and methyl methanesulfonate (MMS), but its sensitivity is milder than that of the rhp51 single mutant [10,13]. The swi5 rhp51 double mutant is as sensitive as the rhp51 single mutant to these DNA damaging treatments. These epistatic relationships suggest that Swi5 functions in an Rhp51-dependent recombinational repair pathway [13]. Both the swi5 and sfr1 single mutants have very similar DNA repair defects each other, and the swi5 sfr1 double mutant has almost the same damage sensitivity as each single mutant. In addition, the swi5 rhp51 double mutant and the swi5 sfr1 rhp51 triple mutant have the same damage sensitivity as the rhp51 single mutant. These results provide rigorous genetic evidence that the Swi5–Sfr1 complex functions in the Rhp51-dependent recombination pathway [13]. Previous works have shown that the Rhp55–Rhp57 heterodimer is implicated in the Rhp51-dependent recombination pathway [21–23]. S. pombe Rhp55 and Rhp57 are counterparts of S. cerevisiae Rad55 and Rad57, respectively, and
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they all are referred to as Rad51 paralogues because of their amino acid sequence similarity [21,22,24]. Various genetic and cytological studies suggest that the Rad55–Rad57 heterodimer functions in the Rad51-dependent recombination pathway as an accessory factor for Rad51 (reviewed in [25]). P. Sung biochemically demonstrated that the S. cerevisiae Rad55–Rad57 heterodimer assists Rad51 nucleoprotein filament formation in vitro, suggesting to act as a recombination mediator [26]. Therefore, the Rhp55–Rhp57 heterodimer is also implicated in the similar mediator function. What is the relationship between the two Rhp51dependent recombination sub-pathways that involve Swi5–Sfr1 complex and the Rhp55–Rhp57 heterodimer? Genetic analysis clearly indicates their relationships during recombinational repair. Each of the rhp55, rhp57, swi5 and sfr1 single mutants shows modest sensitivity to DNA damaging agents such as UV, ␥-ray and MMS, with rhp55 and rhp57 slightly more sensitive than swi5 and sfr1. The swi5 rhp57 and sfr1 rhp57 double mutants and the swi5 rhp57 rhp51 triple mutant show the very similar sensitivity as the rhp51 single mutant. Overproduction of Rhp57 does not suppress the swi5 or sfr1 mutation, and overproduction of Swi5 and/or Sfr1 does not suppress the rhp57 mutation. Taken together, these results indicate that Rhp51-dependent recombination has two parallel subpathways, one that is Rhp55–Rhp57-dependent and another that is Swi5–Sfr1 dependent [13].
5. Swi5–Sfr1 and Rhp55–Rhp57 function redundantly in Rhp51 assembly on DNA A recent cytological study of formation of Rhp51 foci after DNA damage shows that Swi5–Sfr1 is involved in Rhp51 assembly at damaged sites [19]. Almost all wild-type cells exhibited significant Rhp51 focal signals in nuclei, with about half of the nuclei having very strong signals, at a relatively high dose of UV irradiation (200 J/m2 ). In the same experimental condition, the frequency of swi5 cells with intense nuclear Rhp51 foci was strongly reduced and the frequency of the cell with weak Rhp51 focal signals are increased; then, the total frequency of cells with Rhp51 signals was only reduced in swi5 cells. Cells with the rhp57 mutation also showed a reduction in Rhp51 assembly that was much milder than what is seen in swi5 cells. The swi5 rhp57 double mutant is more severely affected than either single mutant. In these cells, only a few nuclei with strong Rhp51 foci have been observed although they can partially form weak Rhp51 foci. These results suggest that Swi5 and Rhp57 facilitate or stabilize Rhp51 nucleoprotein filament formation in a redundant manner (Fig. 1) [13,19].
6.
Purification of the Swi5–Sfr1 complex
We found that production of bacterially expressed Sfr1 protein was very poor while recombinant Swi5 alone was efficiently expressed in E. coli. Even worse, the small amount of Sfr1 produced was found to be insoluble. However, co-expression of Swi5 and Sfr1 allowed for a simple purification, in which Sfr1
5
could be overexpressed as a stable complex with Swi5 and the complex was recovered in a soluble fraction. The two proteins were co-purified during all purification steps and the complex can keep an oligomeric form in a solution at very high salt concentrations such as in 1.5 M NaCl [20]. The purified Swi5–Sfr1 complex has ssDNA- and dsDNAbinding activity, although it has no apparent preference for ssDNA or dsDNA. Since Swi5 alone does not show DNA binding activity, Sfr1 is assumed to have a DNA binding domain(s) (unpublished data).
7. The Swi5–Sfr1 complex stimulates Rhp51-dependent strand exchange It has been reported that RPA stimulates DNA strand exchange with long (plasmid-sized) DNA substrates, which can be mediated by from budding yeast or human Rad51 ([27,28], reviewed in [29]). However, the effect of RPA is very subtle in the case of reactions mediated by S. pombe proteins: the intermediates and products of the strand exchange reaction mediated by Rhp51 are almost undetectable, whether carried out in the presence or absence of RPA. In turn, the addition of the Swi5–Sfr1 complex greatly enhances the DNA strand exchange reaction [20]. The optimal concentration is only about 1/20 to 1/10 the concentration of Rhp51 and excess concentrations of Swi5–Sfr1 inhibit the reaction. Note that this reaction still requires RPA. Generally, Rad51-mediated strand-exchange requires ATP, and the S. pombe Rhp51-mediated reaction in the presence of Swi5–Sfr1 is no exception. Since the Swi5–Sfr1 complex alone does not have DNA-strand exchange activity or ATPase activity, the Swi5–Sfr1 complex is suggested to stimulate the Rhp51-dependent strand exchange reaction, but not to promote strand exchange by itself. However, it remains to be determined whether the Swi5–Sfr1 complex is a bona fide mediator, as discussed below.
8. The mechanism of Rhp51-dependent strand exchange stimulated by the Swi5–Sfr1 complex Rad51 from budding yeast and from human cells has an ATPase activity that is greatly enhanced by ssDNA: in the absence of ssDNA and in the presence of dsDNA, their ATPase is very low [30,31]. However, S. pombe Rhp51 has a DNA-independent ATPase activity, which is only modestly enhanced by ssDNA or dsDNA [20,32]. The Swi5–Sfr1 complex enhances the Rhp51 ATPase activity in the presence of ssDNA by two-to three-fold, although it does not affect the activity in the presence of dsDNA or in the absence of DNA. Interestingly, the Swi5–Sfr1 complex does not quantitatively affect the ssDNA binding activity of Rhp51. The binding of Rhp51 is almost saturated at three nucleotides per Rad51 monomer in either the absence or presence of Swi5–Sfr1. Therefore, the Swi5–Sfr1complex does not simply increase the ssDNA binding by Rhp51, but rather it somehow alters the Rhp51 filament, which is referred to as “activation” in many biological reactions.
6
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Is the Swi5–Sfr1 complex, a canonical recombination mediator? The assessment of the mediator activity of Swi5–Sfr1 is, in very real sense, complicated since the Rhp51-mediated strand exchange reaction greatly depends on Swi5–Sfr1 under experimental conditions. When RPA is added later, which is experimentally considered to reduce its inhibitory effect, the reaction still requires Swi5–Sfr1. Therefore, it is difficult to evaluate the overall RPA inhibitory effect on Rhp51-mediated strand exchange reaction in the absence of Swi5–Sfr1. However, when RPA is added to ssDNA first, the product yield is greatly reduced. In this case, long incubation with Rhp51 and Swi5–Sfr1 before starting the reaction gradually restores strand exchange, while the addition of Swi5–Sfr1 after a long incubation of Rph51 does not. These properties indicate that Swi5–Sfr1 has a potential mediator activity [20].
9. Function of the Swi5–Sfr1 complex in meiosis S. cerevisiae Sae3 and Mei5 are Swi5 and Sfr1 homologues, respectively. Sae3 shares sequence homology with the whole region of Swi5 (Swi5 versus Sae3 24%/53% for identical/similar amino acids), while high sequence homology is shared only between the Sfr1 and Mei5 C-terminal regions (Sfr1 versus Mei5 24%/47% for identical/similar amino acids). In addition, overall sequence homologies are relatively low [13,33,34]. Sae3 and Mei5 are expressed only during meiosis and seems to be specific for meiotic recombination associated with a meiosis-specific recombinase Dmc1 [33,34]. The sae3 and mei5 mutants both show phenotypes very similar to that of the dmc1 mutant, which includes reductions in sporulation, spore viability and cross over recombination. Dmc1, Sae3 and Mei5 forms interdependently foci and colocalize on chromosomes in early steps of meiotic division. Taken together, Sae3 and Mei4 are implicated to form complex with Dmc1 and to stimulate Dmc1 assembly to the DSB sites [33,34]. By an analogy to the mitotic function of the S. cerevisiae homologue, S. pombe Swi5 is also expected to act as the mediator for the meiotic-specific recombinase, Dmc1. However, swi5 mutants exhibit severer defects in spore viability, meiotic recombination frequency and the DSB repair than dmc1 mutants, indicating that swi5 is epistatic to dmc1 [14,35]. Thus, Swi5 obviously has the additional function. The rhp55 swi5 and rhp57 swi5 double mutants drastically reduce their spore viability, implying a redundant function between Rhp55–Rhp57 heterodimer and Swi5 [14]. An idea that Swi5 is directly involved in presynaptic formations of both Rhp51 and Dmc1 is well in accordance with the above observation (Fig. 1). What is a meiotic partner with Swi5? A swi2 mutant has no reduction in meiotic recombination [36]. On the other hand, sfr1 has also been identified as mug13, a mutant exhibiting aberrant chromosome segregation in meiosis [37]. The sfr1 mutant reduces meiotic recombination frequency (our unpublished data). In addition, we have shown that the purified Swi5–Sfr1 complex interacts with Dmc1 in an Sfr1-dependent manner [20]. Thus, it is concluded that Sfr1 partners with Swi5 in meiosis as well.
10. The Swi5–Sfr1 complex also stimulates Dmc1-dependent strand exchange in vitro Indeed, the Swi5–Sfr1 complex was found to stimulate Dmc1mediated strand exchange in much the similar way that it affects the Rhp51-mediated strand exchange reaction [20]. In contrast to its effect on Rhp51, interestingly, it enhanced the ssDNA binding activity of Dmc1. Dmc1 has no, or very little, ATPase activity in the absence of DNA and its ATPase activity requires ssDNA that be bound by Dmc1. The ssDNAdependent ATPase activity of Dmc1 is inhibited by RPA, and Swi5–Sfr1 restores the activity, suggesting it overcomes the RPA inhibitory effect [20]. Although we did not directly demonstrate this in a previous paper, we have recently found that Swi5–Sfr1 stimulates Dmc1-mediated strand exchange using ssDNA pre-coated with RPA under certain experimental conditions (unpublished data). This finding indicates that Swi5–Sfr1 assists in loading Dmc1 onto ssDNA prebound with RPA. In other words, the Swi5–Sfr1 is a canonical mediator at least for Dmc1.
11. Functional differences among Rad22, Rhp55–Rhp57 and Swi5–Sfr1 mediators Another S. pombe mediator is Rad22, a homologue of Rad52 proteins in other eukaryotes including, budding yeast and humans. Genetic studies have indicated that Rad22 is involved in each of two parallel Rhp51-dependent sub-pathways during recombinational repair in S. pombe [38,39]. Therefore, a pair of two mediators (Rad22/Rhp55–Rhp57 and Rad22/Swi5–Sfr1) works in each sub-pathway. What are the functional differences among these mediators? Why are two mediators required in each pathway? A clue comes from our recent biochemical studies (unpublished data). A low efficiency of an Rhp51–Swi5–Sfr1-mediated strand exchange reaction, in which ssDNA is preincubated with RPA, is greatly recovered by the addition of Rad22. On the other hand, Rad52 from budding yeast and humans has been demonstrated to act as a Rad51 mediator, leading to a high yield of strand exchange products [40–43]. However, S. pombe Rad22 alone does not stimulate the Rhp51-mediated strand exchange. Swi5–Sfr1 still requires a full Rhp51-mediated strand exchange reaction. These results suggest that the molecular roles of the two mediators are different, and that both are essential for full Rhp51-mediated strand exchange activity (unpublished data). A working hypothesis is as follows: Rad22 is recruited onto RPA-coated ssDNA regions via a high affinity for RPA, allowing loading of inactive Rhp51. Subsequently, Swi5–Sfr1 activates the Rhp51, which promotes to extend and stabilize Rhp51 nucleofilament with concomitant disassembly of RPA from ssDNA. We assume that Rhp55–Rhp57 plays a biochemical role similar to that of Swi5–Sfr1 in Rhp51-dependent strand exchange. Further biochemical studies will be necessary to understand the molecular function of Rhp55–Rhp57 in the context of Rad22 mediator-dependent HR pathway. How do the in vivo functions of Rhp55–Rhp57 and Swi5–Sfr1 differ? We examined products generated by HO endonucleaseinduced DSBs in these mutant cells, an assay that was
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7
Fig. 3 – A model for a action of Swi5–Sfr1 on the two mediators. Rhp51 loads onto ssDNA by itself as an inactive filament and the Swi5–Sfr1 complex activates the Rhp51 filament. On the other hand, the Swi5–Sfr1 mediator directly assists Dmc1 to load onto ssDNA. The resultant Dmc1 filament that contains Swi5–Sfr1 is an active form.
originally developed by the Humphrey group [44]. Canonical crossover recombinational repair products could be detected at low but significant frequencies in wild-type, swi5 and sfr1 strains, while they were not detected in rhp57 and rhp51 single mutants or in the rhp57 swi5 double mutant, suggesting that Rhp51 and Rhp57, but not Swi5–Sfr1, are essential for crossover production. These results suggest that Swi5–Sfr1 processes DSBs in a manner different from that of the Rhp55–57 mediator (Fig. 1) [19]. However, at about the same time, the Freyer group independently reported that the rhp57 cells produce more cross over products than wild type cells in the same HO-induced DSB repair assay [45]. The reason for this discrepancy is unclear.
12.
Perspective
The apparent effects of the Swi5–Sfr1 accessory factor with respect to Rhp51 and Dmc1 are different. However, this may be only one point of view, and the fundamental contribution of Swi5–Sfr1 to the two recombinases could be the same: to promote the formation of an active nucleoprotein filament (Fig. 3). The differences between the two recombinases might be due to differences in their abilities to promote filament formation. Rhp51 alone has the potential to form a nucleoprotein filament, but Dmc1 does not. Dmc1 easily forms rings or stacked-rings in the presence of DNA and does not readily form helical filaments. Swi5–Sfr1 assists in the formation of the active helical filament from the Dmc1 stacked structure, and the helical filament structure of Dmc1 is the active form for strand exchange. On the other hand, because Rhp51 has the potential to form a nucleoprotein filament by itself,
and Swi5–Sfr1 may serve only to activate the inactive Rhp51 filament. A central question concerns why both recombinases are required in meiosis. The fundamental biochemical functions of Rad51 and Dmc1 are very similar and there are no significant differences between them, at least based our biochemical knowledge of each protein. Many accessory factors could control each recombinase to work cooperatively in a spatial and temporal manner. Swi5–Sfr1 is a strong candidate as a key to understanding the control mechanisms of these two recombinases in meiosis. Swi5 forms a protein complex with Swi2, which participates in MT switching, a gene conversion event not associated with crossover. It has been speculated that Swi5–Swi2 also functions as a mediator in the MT switching reaction, which also involves Rhp51. Determination of further biochemical functions of Swi2 will shed light on a role of Swi5 in influencing homologous recombination and mating type switching. In conclusion, Swi5 is a newly identified global regulator in DNA rearrangement reactions that involve RecA-type recombinases. Molecular analysis of DNA rearrangement mechanisms involving Swi5 has just started.
Acknowledgements Our study was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology (MECSST) of Japan and from the Japan Society for the Promotion of Science (JSPS), and by a grant for the 2005 Strategic Research Project (no. K17005) of Yokohama City University. NH was supported by JSPS fellowships for young scientists.
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