Incompatibility with Formin Cdc12p Prevents Human Profilin from Substituting for Fission Yeast Profilin: INSIGHTS FROM CRYSTAL STRUCTURES OF FISSION YEAST PROFILIN*S?

Expression of human profilin-I does not complement the temperature-sensitive cdc3-124 mutation of the single profilin gene in fission yeast Schizosaccharomyces pombe, resulting in death from cytokinesis defects. Human profilin-I and S. pombe profilin have similar affinities for actin monomers, the FH1 domain of fission yeast formin Cdc12p and poly-l-proline (Lu, J., and Pollard, T. D. (2001) Mol. Biol. Cell 12, 1161–1175), but human profilin-I does not stimulate actin filament elongation by formin Cdc12p like S. pombe profilin. Two crystal structures of S. pombe profilin and homology models of S. pombe profilin bound to actin show how the two profilins bind to identical surfaces on animal and yeast actins even though 75% of the residues on the profilin side of the interaction differ in the two profilins. Overexpression of human profilin-I in fission yeast expressing native profilin also causes cytokinesis defects incompatible with viability. Human profilin-I with the R88E mutation has no detectable affinity for actin and does not have this dominant overexpression phenotype. The Y6D mutation reduces the affinity of human profilin-I for poly-l-proline by 1000-fold, but overexpression of Y6D profilin in fission yeast is lethal. The most likely hypotheses to explain the incompatibility of human profilin-I with Cdc12p are differences in interactions with the proline-rich sequences in the FH1 domain of Cdc12p and wider “wings” that interact with actin.The small protein profilin not only helps to maintain a cytoplasmic pool of actin monomers ready to elongate actin filament barbed ends (2), but it also binds to type II poly-l-proline helices (3, 4). The actin (5) and poly-l-proline (6–8) binding sites are on opposite sides of the profilin molecule, so profilin can link actin to proline-rich targets. Viability of fission yeast depends independently on profilin binding to both actin and poly-l-proline, although cells survive >10-fold reductions in affinity for either ligand (1).Fission yeast Schizosaccharomyces pombe depend on formin Cdc12p (9, 10) and profilin (11) to assemble actin filaments for the cytokinetic contractile ring. Formins are multidomain proteins that nucleate and assemble unbranched actin filaments (12). Formin FH2 domains form homodimers that can associate processively with the barbed ends of growing actin filaments (13, 14). FH2 dimers slow the elongation of barbed ends (15). Most formin proteins have an FH1 domain linked to the FH2 domain. Binding profilin-actin to multiple polyproline sites in an FH1 domain concentrates actin near the barbed end of an actin filament associated with a formin FH2 homodimer. Actin transfers very rapidly from the FH1 domains onto the filament end (16) allowing profilin to stimulate elongation of the filament (15, 17).We tested the ability of human (Homo sapiens, Hs)⁷ profilin-I to complement the temperature-sensitive cdc3-124 mutation (11) in the single fission yeast profilin gene with the aim of using yeast to characterize human profilin mutations. The failure of expression of Hs profilin-I to complement the cdc3-124 mutation prompted us to compare human and fission yeast profilins more carefully. We report here a surprising incompatibility of Hs profilin-I with fission yeast formin Cdc12p, a crystal structure of fission yeast profilin, which allowed a detailed comparison with Hs profilin, and mutations that revealed how overexpression of Hs profilin-I compromises the viability of wild-type fission yeast.     

The Mei5-Sae3 Protein Complex Mediates Dmc1 Activity in Saccharomyces cerevisiae

During homologous recombination, a number of proteins cooperate to catalyze the loading of recombinases onto single-stranded DNA. Single-stranded DNA-binding proteins stimulate recombination by coating single-stranded DNA and keeping it free of secondary structure; however, in order for recombinases to load on single-stranded-DNA-binding protein-coated DNA, the activity of a class of proteins known as recombination mediators is required. Mediator proteins coordinate the handoff of single-stranded DNA from single-stranded DNA-binding protein to recombinase. Here we show that a complex of Mei5 and Sae3 from Saccharomyces cerevisiae preferentially binds single-stranded DNA and relieves the inhibition of the strand assimilation and DNA binding abilities of the meiotic recombinase Dmc1 imposed by the single-stranded DNA-binding protein replication protein A. Additionally, we demonstrate the physical interaction of Mei5-Sae3 with replication protein A. Our results, together with previous in vivo studies, indicate that Mei5-Sae3 is a mediator of Dmc1 assembly during meiotic recombination in S. cerevisiae.During meiosis, recombination between homologous chromosomes ensures proper segregation into haploid products. Recombination events are initiated by the formation of double strand breaks (DSBs)² in DNA (1). This is followed by resection of free DNA ends to yield 3′ single-stranded tails, upon which recombinase assembles to form nucleoprotein filaments. Following recombinase assembly, the nucleoprotein filament engages a donor chromatid, searches for homologous DNA sequences on that chromatid, and promotes strand exchange to yield a heteroduplex DNA intermediate often referred to as a joint molecule. Although recombinase alone is capable of promoting homology search and strand exchange in vitro, genetic and biochemical studies have demonstrated that normal recombinase function in vivo requires the activity of a number of accessory factors (2). These factors enhance the assembly of nucleoprotein filaments, target capture, homology search, and dissociation of recombinase from duplex DNA.Most eukaryotes possess two recombinases, both homologues of the Escherichia coli recombinase RecA: Rad51, which is the major recombinase in mitotic cells and is also important during meiotic recombination, and Dmc1, which functions only in meiosis. Dmc1 and Rad51 have been shown to assemble at DSBs by immunofluorescence and chromatin immunoprecipitation (3–6), and both proteins oligomerize on single-stranded DNA (ssDNA) to form nucleofilaments that catalyze strand invasion (7–9).A number of biochemical studies have defined the role of accessory factors in stimulating the activity of Rad51 (10–12). Replication protein A (RPA), the yeast ssDNA-binding protein (SSB), removes secondary structure in ssDNA that otherwise prevents formation of fully functional nucleoprotein filaments (13). Both Rad52 protein (11, 12) and the heterodimeric protein Rad55/Rad57 (14) can overcome the inhibitory effect of RPA on Rad51 nucleoprotein filament formation in purified systems, mediating a handoff between RPA and Rad51. It is thought that the mechanism for the mediator activity of Rad52 involves Rad52 recognizing and binding to RPA-coated ssDNA, where it provides nucleation sites for the recruitment of free molecules of Rad51 (15). The tumor suppressor protein BRCA2 also serves as an assembly factor for Rad51 during mitosis in a variety of species that encode orthologues of this protein, including mice (16), corn smut (17), and humans (18).The meiosis-specific recombinase Dmc1 is stimulated by a distinct set of accessory factors. Immunostaining studies suggest that the Rad51 mediators Rad52 and Rad55/Rad57 are not required for assembly of Dmc1 foci in vivo, although Rad51 itself promotes Dmc1 foci (19–21). More recently, immunostaining and chromatin immunoprecipitation experiments demonstrated a role for the Mei5 and Sae3 proteins of Saccharomyces cerevisiae in assembly of Dmc1 at sites of DSBs in vivo (22, 23). Consistent with these observations, mei5 and sae3 mutants display markedly similar meiotic defects as compared with dmc1 mutants, including defects in sporulation, spore viability, crossing over, DSB repair, progression through meiosis, and synaptonemal complex formation (19, 22–24). Finally, the three proteins have been shown to physically interact; Mei5 and Sae3 have been co-purified and co-immunoprecipitated, and an N-terminal portion of Mei5 has been shown to interact with Dmc1 in a two-hybrid assay (22).The fission yeast Schizosaccharomyces pombe encodes two proteins, Swi5 and Sfr1, which share sequence homology with Sae3 and Mei5, respectively (22). Swi5 and Sfr1 have been shown to stimulate the strand exchange activity of Rhp51 (the S. pombe Rad51 homologue) and Dmc1 (25). Although some results indicate functional similarity of Swi5-Sfr1 and Mei5-Sae3, there are also clear differences. The Mei5-Sae3 complex of budding yeast is expressed solely during meiosis, and no mitotic phenotypes have been reported for mei5 or sae3 mutants (22, 24, 26). In contrast, the Swi5-Sfr1 complex of fission yeast is expressed in mitotic and meiotic cells, and mutations in SWI5 have been shown to cause defects in mitotic recombination (27). Furthermore, although mei5 and sae3 mutants are phenotypically similar to dmc1 mutants, swi5 and sfr1 mutants display more severe meiotic defects during fission yeast meiosis than do dmc1 mutants (27–29). These data suggest that although Swi5-Sfr1 clearly contributes to Rad51 activity in fission yeast, it is possible that the activity of Mei5-Sae3 is restricted to stimulating Dmc1 in budding yeast.In this study, a biochemical approach is used to test the budding yeast Mei5-Sae3 complex for properties expected of a recombinase assembly mediator. We show that Mei5-Sae3 binds both ssDNA and double-stranded DNA (dsDNA) but binds ssDNA preferentially. We also show that Mei5-Sae3 can overcome the inhibitory effects of RPA on the ssDNA binding and strand assimilation activities of Dmc1. Finally, we show that Mei5-Sae3 and RPA bind one another directly. These results indicate that Mei5-Sae3 acts directly as a mediator protein for assembly of Dmc1.     

Intersectin Regulates Dendritic Spine Development and Somatodendritic Endocytosis but Not Synaptic Vesicle Recycling in Hippocampal Neurons

Intersectin-short (intersectin-s) is a multimodule scaffolding protein functioning in constitutive and regulated forms of endocytosis in non-neuronal cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of Drosophila and Caenorhabditis elegans. In vertebrates, alternative splicing generates a second isoform, intersectin-long (intersectin-l), that contains additional modular domains providing a guanine nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is expressed in multiple tissues and cells, including glia, but excluded from neurons, whereas intersectin-l is a neuron-specific isoform. Thus, intersectin-I may regulate multiple forms of endocytosis in mammalian neurons, including SV endocytosis. We now report, however, that intersectin-l is localized to somatodendritic regions of cultured hippocampal neurons, with some juxtanuclear accumulation, but is excluded from synaptophysin-labeled axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV recycling. Instead intersectin-l co-localizes with clathrin heavy chain and adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces the rate of transferrin endocytosis. The protein also co-localizes with F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation during development. Our data indicate that intersectin-l is indeed an important regulator of constitutive endocytosis and neuronal development but that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis (CME)⁴ is a major mechanism by which cells take up nutrients, control the surface levels of multiple proteins, including ion channels and transporters, and regulate the coupling of signaling receptors to downstream signaling cascades (1-5). In neurons, CME takes on additional specialized roles; it is an important process regulating synaptic vesicle (SV) availability through endocytosis and recycling of SV membranes (6, 7), it shapes synaptic plasticity (8-10), and it is crucial in maintaining synaptic membranes and membrane structure (11).Numerous endocytic accessory proteins participate in CME, interacting with each other and with core components of the endocytic machinery such as clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific modules and peptide motifs (12). One such module is the Eps15 homology domain that binds to proteins bearing NPF motifs (13, 14). Another is the Src homology 3 (SH3) domain, which binds to proline-rich domains in protein partners (15). Intersectin is a multimodule scaffolding protein that interacts with a wide range of proteins, including several involved in CME (16). Intersectin has two N-terminal Eps15 homology domains that are responsible for binding to epsin, SCAMP1, and numb (17-19), a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25 (17, 20, 21), and five SH3 domains in its C-terminal region that interact with multiple proline-rich domain proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS (16, 22-25). The rich binding capability of intersectin has linked it to various functions from CME (17, 26, 27) and signaling (22, 28, 29) to mitogenesis (30, 31) and regulation of the actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of Drosophila and C. elegans where it acts as a scaffold, regulating the synaptic levels of endocytic accessory proteins (21, 32-34). In vertebrates, the intersectin gene is subject to alternative splicing, and a longer isoform (intersectin-l) is generated that is expressed exclusively in neurons (26, 28, 35, 36). This isoform has all the binding modules of its short (intersectin-s) counterpart but also has additional domains: a DH and a PH domain that provide guanine nucleotide exchange factor (GEF) activity specific for Cdc42 (23, 37) and a C2 domain at the C terminus. Through its GEF activity and binding to actin regulatory proteins, including N-WASP, intersectin-l has been implicated in actin regulation and the development of dendritic spines (19, 23, 24). In addition, because the rest of the binding modules are shared between intersectin-s and -l, it is generally thought that the two intersectin isoforms have the same endocytic functions. In particular, given the well defined role for the invertebrate orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l performs this role in mammalian neurons, which lack intersectin-s. Defining the complement of intersectin functional activities in mammalian neurons is particularly relevant given that the protein is involved in the pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is localized on chromosome 21q22.2 and is overexpressed in DS brains (38). Interestingly, alterations in endosomal pathways are a hallmark of DS neurons and neurons from the partial trisomy 16 mouse, Ts65Dn, a model for DS (39, 40). Thus, an endocytic trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured hippocampal neurons. We find that intersectin-l is localized to the somatodendritic regions of neurons, where it co-localizes with CHC and AP-2 and regulates the uptake of transferrin. Intersectin-l also co-localizes with actin at dendritic spines and disrupting intersectin-l function alters dendritic spine development. In contrast, intersectin-l is absent from presynaptic terminals and has little or no role in SV recycling.     

Determination of in Vivo Dissociation Constant, KD, of Cdc42-Effector Complexes in Live Mammalian Cells Using Single Wavelength Fluorescence Cross-correlation Spectroscopy

The RhoGTPase Cdc42 coordinates cell morphogenesis, cell cycle, and cell polarity decisions downstream of membrane-bound receptors through distinct effector pathways. Cdc42-effector protein interactions represent important elements of cell signaling pathways that regulate cell biology in systems as diverse as yeast and humans. To derive mechanistic insights into cell signaling pathways, it is vital that we generate quantitative data from in vivo systems. We need to be able to measure parameters such as protein concentrations, rates of diffusion, and dissociation constants (K_D) of protein-protein interactions in vivo. Here we show how single wavelength fluorescence cross-correlation spectroscopy in combination with Förster resonance energy transfer analysis can be used to determine K_D of Cdc42-effector interactions in live mammalian cells. Constructs encoding green fluorescent protein or monomeric red fluorescent protein fusion proteins of Cdc42, an effector domain (CRIB), and two effectors, neural Wiskott-Aldrich syndrome protein (N-WASP) and insulin receptor substrate protein (IRSp53), were expressed as pairs in Chinese hamster ovary cells, and concentrations of free protein as well as complexed protein were determined. The measured K_D for Cdc42V12-N-WASP, Cdc42V12-CRIB, and Cdc42V12-IRSp53 was 27, 250, and 391 nm, respectively. The determination of K_D for Cdc42-effector interactions opens the way to describe cell signaling pathways quantitatively in vivo in mammalian cells.Over the last 2 decades, we have been successful in describing a myriad of cell signaling pathways that regulate the biology of cells. These pathways are made of elements incorporating protein-protein, protein-lipid and protein-ligand interactions. With the advent of GFP² (1, 2) and its variants (3), it is now possible to genetically encode fluorescent probes into any protein of interest. GFP fusion proteins can be used in live cells giving spatial and temporal resolution to cell signaling pathways (4). To gain mechanistic insights into cellular processes, it is crucial that we measure quantitative parameters to describe cell signaling. In this study, we present an approach based on fluorescence cross-correlation spectroscopy (FCCS) (5, 6) and Förster resonance energy transfer (FRET) to determine quantitative parameters of cell signaling pathways, including the determination of the K_D for Cdc42-effector interactions in live CHO-K-1 (hereafter referred to as CHO) mammalian cells.The RhoGTPase Cdc42 (7, 8) regulates pathways that coordinate cell cycle, morphogenesis, and polarity. Cdc42 is a molecular switch that cycles between an inactive (GDP-bound) and active (GTP-bound) state. The V12 Cdc42 point mutation freezes the protein in an activated GTP-bound form, which binds effectors strongly. In contrast, Cdc42N17 is a dominant negative protein that is GDP-bound and interacts with effectors weakly if at all (9). A major Cdc42 binding site/domain in effector proteins is known as Cdc42- and Rac-interacting binding region (CRIB)³ and was originally found in activated Cdc42 kinase, p21 activated kinase (PAK), and neural Wiskott-Aldrich syndrome protein (N-WASP) (10). The inverse Bin-amphiphysins-Rvs domain adaptor protein IRSp53 is also an effector but binds Cdc42 through a partial CRIB domain (11, 12). Cdc42 interaction with its effectors has two main consequences, which are not mutually exclusive: (i) unfolding of effector to expose the active site and (ii) relocalization of effector to membrane compartments. Thus Cdc42-effector interactions serve as a good model for cell signaling as a whole.Fluorescence correlation spectroscopy and FCCS measure fluctuations in fluorescence of a small number of molecules as they pass through a defined confocal volume, respectively (13, 14, 15). Since the number of molecules in the confocal volume and the confocal volume itself can be determined, concentrations of protein can be measured by fluorescence correlation spectroscopy. Single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS) is an FCCS variant in which excitation of two or more probes is achieved by single wavelength one-photon excitation. To date SW-FCCS has been used successfully to follow receptors and receptor-ligand interactions in vitro and in vivo (6, 16, 17).In the present analysis, we take a two-step approach to determining the K_D of Cdc42 binding to CRIB (domain of PAK), N-WASP, and IRSp53. First, we show that the proteins under investigation are indeed interacting with each other directly in vivo by FRET analysis. Here we use acceptor photobleaching (AP)-FRET as well as changes in lifetime (through fluorescence lifetime imaging microscopy (FLIM)) as indicators of FRET. Second, we use SW-FCCS to determine the K_D of Cdc42 interacting with its effectors by measuring the concentration of free protein versus complexed protein. Thus, the combined use of FRET and FCCS allows quantitative analysis of cell signaling pathways in vivo.     

Phosphorylation of p53 Is Regulated by TPX2-Aurora A in Xenopus Oocytes

p53 is an important tumor suppressor regulating the cell cycle at multiple stages in higher vertebrates. The p53 gene is frequently deleted or mutated in human cancers, resulting in loss of p53 activity. This leads to centrosome amplification, aneuploidy, and tumorigenesis, three phenotypes also observed after overexpression of the oncogenic kinase Aurora A. Accordingly, recent studies have focused on the relationship between these two proteins. p53 and Aurora A have been reported to interact in mammalian cells, but the function of this interaction remains unclear. We recently reported that Xenopus p53 can inhibit Aurora A activity in vitro but only in the absence of TPX2. Here we investigate the interplay between Xenopus Aurora A, TPX2, and p53 and show that newly synthesized TPX2 is required for nearly all Aurora A activation and for full p53 synthesis and phosphorylation in vivo during oocyte maturation. In vitro, phosphorylation mediated by Aurora A targets serines 129 and 190 within the DNA binding domain of p53. Glutathione S-transferase pull-down studies indicate that the interaction occurs via the p53 transactivation domain and the Aurora A catalytic domain around the T-loop. Our studies suggest that targeting of TPX2 might be an effective strategy for specifically inhibiting the phosphorylation of Aurora A substrates, including p53.Aurora A is an oncogenic protein kinase that is active in mitosis and plays important roles in spindle assembly and centrosome function (1). Overexpression of either human or Xenopus Aurora A transforms mammalian cells, but only when the p53 pathway is altered (2–4). Aurora A is localized on centrosomes during mitosis, and overexpression of the protein leads to centrosome amplification and aneuploidy (2, 3, 5, 6), two likely contributors to genomic instability (7, 8). Because of its oncogenic potential and amplification in human tumors, considerable attention has been focused on the mechanism of Aurora A activation in mitosis. Evidence from several laboratories indicates that activation occurs as a result of phosphorylation of a threonine residue in the T-loop of the kinase (4, 9, 10). Purification of Aurora A-activating activity from M phase Xenopus egg extracts led to an apparent activation mechanism in which autophosphorylation at the T-loop is stimulated by binding of the targeting protein for Xklp2 (TPX2) (11–14). On the other hand, it has been shown that Aurora A activity can be inhibited by interaction with several proteins, including PP1 (protein phosphatase 1), AIP (Aurora A kinase-interacting protein), and, more recently, p53 (9, 15–17).p53 is a well known tumor suppressor able to drive cell cycle arrest, apoptosis, or senescence when DNA is damaged or cell integrity is threatened (18, 19). In human cancers, the p53 gene is frequently deleted or mutated, leading to inactivation of p53 functions (20). p53 protein is almost undetectable in “normal cells,” mainly due to its instability. Indeed, during a normal cell cycle, p53 associates with Mdm2 in the nucleus and thereafter undergoes nuclear exclusion, allowing its ubiquitination and subsequent degradation (21). In cells under stress, p53 is stabilized through the disruption of its interaction with Mdm2 (21), leading to p53 accumulation in the nucleus and triggering different responses, as described above.Although p53 has mostly been characterized as a nuclear protein, it has also been shown to localize on centrosomes (22–24) and regulate centrosome duplication (23, 24). Centrosomes are believed to act as scaffolds that concentrate many regulatory molecules involved in signal transduction, including multiple protein kinases (25). Thus, centrosomal localization of p53 might be important for its own regulation by phosphorylation/dephosphorylation, and one of its regulators could be the mitotic kinase Aurora A. Indeed, phenotypes associated with the misexpression of these two proteins are very similar. For example, overexpression of Aurora A kinase leads to centrosome amplification, aneuploidy, and tumorigenesis, and the same effects are often observed after down-regulation of p53 transactivation activity or deletion/mutation of its gene (26, 27).Several recent studies performed in mammalian models show interplay between p53 and Aurora A, with each protein having the ability to inhibit the other, depending on the stage of the cell cycle and the stress level of the cell (17, 28, 29). These studies reported that p53 is a substrate of Aurora A, and serines 215 and 315 were demonstrated to be the two major Aurora A phosphorylation sites in human p53 in vitro and in vivo. Phosphorylation of Ser-215 within the DNA binding domain of human p53 inhibited both p53 DNA binding and transactivation activities (29). Recently, our group showed that Xenopus p53 is able to inhibit Aurora A kinase activity in vitro, but this inhibitory effect can be suppressed by prior binding of Aurora A to TPX2 (9). Contrary to somatic cells, where p53 is nuclear, unstable, and expressed at a very low level, p53 is highly expressed in the cytoplasm of Xenopus oocytes and stable until later stages of development (30, 31). The high concentration of both p53 and Aurora A in the oocyte provided a suitable basis for investigating p53-Aurora A interaction and also evaluating Xenopus p53 as a substrate of Aurora A.     

RecR-mediated Modulation of RecF Dimer Specificity for Single- and Double-stranded DNA

RecF pathway proteins play an important role in the restart of stalled replication and DNA repair in prokaryotes. Following DNA damage, RecF, RecR, and RecO initiate homologous recombination (HR) by loading of the RecA recombinase on single-stranded (ss) DNA, protected by ssDNA-binding protein. The specific role of RecF in this process is not well understood. Previous studies have proposed that RecF directs the RecOR complex to boundaries of damaged DNA regions by recognizing single-stranded/double-stranded (ss/ds) DNA junctions. RecF belongs to ABC-type ATPases, which function through an ATP-dependent dimerization. Here, we demonstrate that the RecF of Deinococcus radiodurans interacts with DNA as an ATP-dependent dimer, and that the DNA binding and ATPase activity of RecF depend on both the structure of DNA substrate, and the presence of RecR. We found that RecR interacts as a tetramer with the RecF dimer. RecR increases the RecF affinity to dsDNA without stimulating ATP hydrolysis but destabilizes RecF binding to ssDNA and dimerization, likely due to increasing the ATPase rate. The DNA-dependent binding of RecR to the RecF-DNA complex occurs through specific protein-protein interactions without significant contributions from RecR-DNA interactions. Finally, RecF neither alone nor in complex with RecR preferentially binds to the ss/dsDNA junction. Our data suggest that the specificity of the RecFOR complex toward the boundaries of DNA damaged regions may result from a network of protein-protein and DNA-protein interactions, rather than a simple recognition of the ss/dsDNA junction by RecF.Homologous recombination (HR)² is one of the primary mechanisms by which cells repair dsDNA breaks (DSBs) and ssDNA gaps (SSGs), and is important for restart of stalled DNA replication (1). HR is initiated when RecA-like recombinases bind to ssDNA forming an extended nucleoprotein filament, referred to as a presynaptic complex (2). The potential for genetic rearrangements dictates that HR initiation is tightly regulated at multiple levels (1). During replication, the ssDNA-binding protein (SSB) protects transiently unwound DNA chains, preventing interactions with recombinases. Following DNA damage, recombination mediator proteins (RMPs) initiate HR by facilitating the formation of the recombinase filaments with ssDNA, while removing SSB (3, 4). Mutations in human proteins involved in HR initiation are linked to cancer predisposition, chromosome instability, UV sensitivity, and premature aging diseases (4–8). To date, little is known about the mechanism by which RMPs regulate the formation of the recombinase filaments on the SSB-protected ssDNA.In Escherichia coli, there are two major recombination pathways, RecBCD and RecF (9, 10). A helicase/nuclease RecBCD complex processes DSBs and recruits RecA on ssDNA in a sequence-specific manner (11–13). The principle players in the RecF pathway are the RecF, RecO, and RecR proteins, which form an epistatic group that is important for SSG repair, for restart of stalled DNA replication, and under specific conditions, can also process DSBs (14–20). Homologs of RecF, -O, and -R are present in the majority of known bacteria (21), including Deinococcus radiodurans, extremely radiation-resistant bacteria that lacks the RecBCD pathway, yet is capable of repairing thousands of DSBs (22, 23). In addition, the sequence or functional homologs of RecF pathway proteins are involved in similar pathways in eukaryotes that include among others WRN, BLM, RAD52, and BRCA2 proteins (4–8).The involvement of all three RecF, -O, and -R proteins in HR initiation is well documented by genetic and cellular approaches (18, 24–30), yet their biochemical functions in the initiation process remain unclear, particularly with respect to RecF. RecO and RecR proteins are sufficient to promote formation of the RecA filament on SSB-bound ssDNA in vitro (27). The UV-sensitive phenotype of recF mutants can be suppressed by RecOR overexpression, suggesting that RecF may direct the RMP complex to DNA-damaged regions where HR initiation is required (31). In agreement with this hypothesis, RecF dramatically increases the efficiency of the RecA loading at ds/ssDNA junctions with a 3′ ssDNA extension under specific conditions (32). RecF and RecR proteins also prevent the RecA filaments from extending into dsDNA regions adjacent to SSGs (33). These data suggest that RecF may directly recognize an ss/dsDNA junction structure (34). However, DNA binding experiments have not provided clear evidence to support such a hypothesis (11).The targeting promoted by RecF may also occur through more complex processes. RecF shares a high structural similarity with the head domain of Rad50, an ABC-type ATPase that recognizes DSBs and initiates repair in archaea and eukaryotes (35). All known ABC-type ATPases function as oligomeric complexes in which a sequence of inter- and intra-molecular interactions is triggered by the ATP-dependent dimerization and the dimer-dependent ATP hydrolysis (36–39). RecF is also an ATP-dependent DNA-binding protein and a weak DNA-dependent ATPase (11, 40). RecF forms an ATP-dependent dimer and all three conserved motifs (Walker A, Walker B, and “signature”) of RecF are important for ATP-dependent dimerization, ATP hydrolysis, and functional resistance to DNA damage (35). Thus, RecF may function in recombination initiation through a complex pathway of protein-protein and DNA-protein interactions regulated by ATP-dependent RecF dimerization.In this report, we present a detailed characterization of the RecF dimerization, and its role in the RecF interaction with various DNA substrates, with RecR, and in ATP hydrolysis. Our data outline the following key findings. First, RecF interacts with DNA as a dimer. Second, neither RecF alone nor the RecFR complex preferentially binds the ss/dsDNA junction. Finally, RecR changes the ATPase activity and the DNA binding of RecF by destabilizing the interaction with ssDNA, and greatly enhancing the interaction with dsDNA. Our results suggest that the specificity of RecF for the boundaries of SSGs is likely to result from a sequence of protein-protein interaction events rather than a simple RecF ss/dsDNA binding, underlining a highly regulated mechanism of the HR initiation by the RecFOR proteins.     

Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL

The RAD51 protein is a central player in homologous recombinational repair. The RAD51B protein is one of five RAD51 paralogs that function in the homologous recombinational repair pathway in higher eukaryotes. In the present study, we found that the human EVL (Ena/Vasp-like) protein, which is suggested to be involved in actin-remodeling processes, unexpectedly binds to the RAD51 and RAD51B proteins and stimulates the RAD51-mediated homologous pairing and strand exchange. The EVL knockdown cells impaired RAD51 assembly onto damaged DNA after ionizing radiation or mitomycin C treatment. The EVL protein alone promotes single-stranded DNA annealing, and the recombination activities of the EVL protein are further enhanced by the RAD51B protein. The expression of the EVL protein is not ubiquitous, but it is significantly expressed in breast cancer-derived MCF7 cells. These results suggest that the EVL protein is a novel recombination factor that may be required for repairing specific DNA lesions, and that may cause tumor malignancy by its inappropriate expression.Chromosomal DNA double strand breaks (DSBs)² are potential inducers of chromosomal aberrations and tumorigenesis, and they are accurately repaired by the homologous recombinational repair (HRR) pathway, without base substitutions, deletions, and insertions (1–3). In the HRR pathway (4, 5), single-stranded DNA (ssDNA) tails are produced at the DSB sites. The RAD51 protein, a eukaryotic homologue of the bacterial RecA protein, binds to the ssDNA tail and forms a helical nucleoprotein filament. The RAD51-ssDNA filament then binds to the intact double-stranded DNA (dsDNA) to form a three-component complex, containing ssDNA, dsDNA, and the RAD51 protein. In this three-component complex, the RAD51 protein promotes recombination reactions, such as homologous pairing and strand exchange (6–9).The RAD51 protein requires auxiliary proteins to promote the homologous pairing and strand exchange reactions efficiently in cells (10–12). In humans, the RAD52, RAD54, and RAD54B proteins directly interact with the RAD51 protein (13–17) and stimulate the RAD51-mediated homologous pairing and/or strand exchange reactions in vitro (18–21). The human RAD51AP1 protein, which directly binds to the RAD51 protein (22), was also found to stimulate RAD51-mediated homologous pairing in vitro (23, 24). The BRCA2 protein contains ssDNA-binding, dsDNA-binding, and RAD51-binding motifs (25–33), and the Ustilago maydis BRCA2 ortholog, Brh2, reportedly stimulated RAD51-mediated strand exchange (34, 35). Most of these RAD51-interacting factors are known to be required for efficient RAD51 assembly onto DSB sites in cells treated with ionizing radiation (10–12).The RAD51B (RAD51L1, Rec2) protein is a member of the RAD51 paralogs, which share about 20–30% amino acid sequence similarity with the RAD51 protein (36–38). RAD51B-deficient cells are hypersensitive to DSB-inducing agents, such as cisplatin, mitomycin C (MMC), and γ-rays, indicating that the RAD51B protein is involved in the HRR pathway (39–44). Genetic experiments revealed that RAD51B-deficient cells exhibited impaired RAD51 assembly onto DSB sites (39, 44), suggesting that the RAD51B protein functions in the early stage of the HRR pathway. Biochemical experiments also suggested that the RAD51B protein participates in the early to late stages of the HRR pathway (45–47).In the present study, we found that the human EVL (Ena/Vasp-like) protein binds to the RAD51 and RAD51B proteins in a HeLa cell extract. The EVL protein is known to be involved in cytoplasmic actin remodeling (48) and is also overexpressed in breast cancer (49). Like the RAD51B knockdown cells, the EVL knockdown cells partially impaired RAD51 foci formation after DSB induction, suggesting that the EVL protein enhances RAD51 assembly onto DSB sites. The purified EVL protein preferentially bound to ssDNA and stimulated RAD51-mediated homologous pairing and strand exchange. The EVL protein also promoted the annealing of complementary strands. These recombination reactions that were stimulated or promoted by the EVL protein were further enhanced by the RAD51B protein. These results strongly suggested that the EVL protein is a novel factor that activates RAD51-mediated recombination reactions, probably with the RAD51B protein. We anticipate that, in addition to its involvement in cytoplasmic actin dynamics, the EVL protein may be required in homologous recombination for repairing specific DNA lesions, and it may cause tumor malignancy by inappropriate recombination enhanced by EVL overexpression in certain types of tumor cells.     

Sgt1 Dimerization Is Required for Yeast Kinetochore Assembly

The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for high-fidelity chromosome transmission during cell division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study, we show that Sgt1 forms homodimers by performing in vitro and in vivo immunoprecipitation and analytical ultracentrifugation analyses. Analyses of the dimerization of Sgt1 deletion proteins showed that the Skp1-binding domain (amino acids 1–211) contains the Sgt1 homodimerization domain. Also, the Sgt1 mutant proteins that were unable to dimerize also did not bind Skp1, suggesting that Sgt1 dimerization is important for Sgt1-Skp1 binding. Restoring dimerization activity of a dimerization-deficient sgt1 mutant (sgt1-L31P) by using the CENP-B (centromere protein-B) dimerization domain suppressed the temperature sensitivity, the benomyl sensitivity, and the chromosome missegregation phenotype of sgt1-L31P. These results strongly suggest that Sgt1 dimerization is required for kinetochore assembly.Spindle microtubules are coupled to the centromeric region of the chromosome by a structural protein complex called the kinetochore (1, 2). The kinetochore is thought to generate a signal that arrests cells during mitosis when it is not properly attached to microtubules, thereby preventing aberrant chromosome transmission to the daughter cells, which can lead to tumorigenesis (3, 4). The kinetochore of the budding yeast Saccharomyces cerevisiae has been characterized thoroughly, genetically and biochemically; thus, its molecular structure is the most well detailed to date. More than 70 different proteins comprise the budding yeast kinetochore, and several of those are conserved in mammals (2).The budding yeast centromere DNA is a 125-bp region that contains three conserved regions, CDEI, CDEII, and CDEIII (5, 6). CDEI is bound by Cbf1 (7–9). CDEIII (25 bp) is essential for centromere function (10) and is the site where CBF3 binds to centromeric DNA. CBF3 contains four proteins: Ndc10, Cep3, Ctf13 (11–18), and Skp1 (17, 18), all of which are essential for viability. Mutations in any of the four CBF3 proteins abolish the ability of CDEIII to bind to CBF3 (19, 20). All of the described kinetochore proteins, except the CDEI-binding Cbf1, localize to kinetochores dependent on the CBF3 complex (2). Therefore, the CBF3 complex is the fundamental structure of the kinetochore, and the mechanism of CBF3 assembly is of major interest.We previously isolated SGT1, the skp1-4 kinetochore-defective mutant dosage suppressor (21). Sgt1 and Skp1 activate Ctf13; thus, they are required for assembly of the CBF3 complex (21). The molecular chaperone Hsp90 is also required for the formation of the Skp1-Ctf13 complex (22). Sgt1 has two highly conserved motifs that are required for protein-protein interaction, the tetratricopeptide repeat (TPR)² (21) and the CS (CHORD protein- and Sgt1-specific) motif. We and others (23–26) have found that both domains are important for the interaction with Hsp90. The Sgt1-Hsp90 interaction is required for the assembly of the core kinetochore complex; this interaction is an initial step in kinetochore assembly (24, 26, 27) that is conserved between yeast and humans (28, 29).In this study, we further characterized the molecular mechanism of this assembly process. We found that Sgt1 forms dimers in vivo, and our results strongly suggest that Sgt1 dimerization is required for kinetochore assembly in budding yeast.