|
Items in cart [0] |
Questions? Call 888-624-8373 |
PAPERBACK Free Resources |
||||||||||||||||
|
||||||||||||||||
The following HTML text is provided to enhance online readability. Many aspects of typography translate only awkwardly to HTML. Please use the page image as the authoritative form to ensure accuracy. ColloquiumThe synaptic activity of HsDmc1, a human recombination protein specific to meiosisR.C.Gupta, E.Golub, B.Bi, and C.M.Radding* Departments of Genetics, and Molecular Biophysics and Biochemistry, and Yale University School of Medicine, New Haven, CT 06520–8005 Human Dmc1 protein, a meiosis-specific homolog of Escherichia coli RecA protein, has previously been shown to promote DNA homologous pairing and strand-exchange reactions that are qualitatively similar to those of RecA protein and Rad51. Human and yeast Rad51 proteins each form a nucleoprotein filament that is very similar to the filament formed by RecA protein. However, recent studies failed to find a similar filament made by Dmc1 but showed instead that this protein forms octameric rings and stacks of rings. These observations stimulated further efforts to elucidate the mechanism by which Dmc1 promotes the recognition of homology. Dmc1, purified to a state in which nuclease and helicase activities were undetectable, promoted homologous pairing and strand exchange as measured by fluorescence resonance energy transfer (FRET). Observations on the intermediates and products, which can be distinguished by FRET assays, provided direct evidence of a three-stranded synaptic intermediate. The effects of helix stability and mismatched base pairs on the recognition of homology revealed further that human Dmc1, like human Rad51, requires the preferential breathing of A·T base pairs for recognition of homology. We conclude that Dmc1, like human Rad51 and E. coli RecA protein, promotes homologous pairing and strand exchange by a “synaptic pathway” involving a three-stranded nucleoprotein intermediate, rather than by a “helicase pathway” involving the separation and reannealing of DNA strands. Two kinds of macromolecular protein structures play prominent roles in homologous genetic recombination: toroids and filaments. Among the toroidal structures, the best understanding of the relation of function to structure exists for Escherichia coli RuvB protein, which forms a hexameric ATPase/helicase. Two such hexameric rings, interacting with RuvA protein, drive the migration of Holliday structures, the 4-fold branched DNA intermediate formed after initial synapsis and processing have occurred. The consequence of this driven migration is the reciprocal exchange of a pair of like strands between two duplex molecules (1). Rings are also formed by the exonuclease of phage λ (2), the Erf protein of phage P22 (3), and human Rad52 protein (4, 5). On the basis of the crystal structure of toroidal λ exonuclease, Kovall and Matthews suggested a mechanistic explanation of the high processivity of this enzyme (2). Some recombination proteins form both rings and filaments: the β protein of phage λ forms large rings and left-handed helical filaments (6). β protein promotes annealing of complementary single strands (7, 8) and migration of a single-stranded branch (9). This protein, which does not hydrolyze ATP, binds more strongly to the product of its annealing activity, which may help to explain how it drives branch migration (10), but there are no correlations of structure with function that help us to understand the role of either the rings or the left-handed filaments. RecT protein, the product of a cryptic prophage in E. coli and a functional homolog of β protein, also forms rings and filaments. The precise nature of the RecT filament is less clear than that of β but has been judged to be helical (11). In this case as well, few clues link structure and function. Among the filamentous structures, the best understanding of the relation of function to structure exists for E. coli RecA protein and human Rad51 protein. Although these proteins form rings as well as right-handed helical filaments (12–14), genetic (15), electronmicroscopic (16), and biochemical evidence (17) indicates strongly that the filament promotes homologous pairing and strand exchange. The role or significance of the rings is unknown. RecA protein was the first-discovered member of a ubiquitous family of proteins found in prokarya, eukarya, and archea. The much-studied filament that is formed by RecA protein on single-stranded DNA promotes a search for homology in duplex DNA and incorporates the homologous duplex. Incorporation of the duplex produces a synaptic nucleoprotein structure containing three homologously aligned strands of DNA in a right-handed helix. In the case of RecA protein, this synaptic structure leads to extensive strand exchange that dissociates one strand of the parental duplex to replace it with the single strand that nucleates formation of the filament (for review, see ref. 18). Solution of the structure of the single strand has shown that the bases, whose axial spacing is 50% greater than in B-form DNA, are stacked alternately with the deoxyribose moieties of the phosphodiester backbone, a configuration that links the angular displacement of bases to a change in the pucker of the sugar (19). Recent studies of human Rad51 indicate that the filament formed on single-stranded DNA provides a catalytic surface, which, on collision with duplex DNA, promotes the opening of base pairs to check for homology at the point of contact anywhere along the filament (20). Dmc1 protein, a true homolog of RecA protein that shares extensive sequence homology with it, is expressed specifically in meiotic cells (21). Discovered in baker’s yeast, it has been found also in mammals (22). Homozygous knockout of Dmc1 in the mouse leads to asynapsis and sterility (23, 24), whereas knockout of Rad51 leads to embryonic lethality (25, 26). Both Dmc1 and Rad51 are essential for meiosis in yeast. In vitro, Dmc1 protein promotes homologous pairing and limited strand exchange (27, 28). On the basis of sequence homology and preliminary biochemical characterization, it seemed likely that Dmc1 protein would catalyze homologous recognition by the same mechanism as that of E. coli RecA and eukaryotic Rad51 proteins. Prominent aspects of this mechanism include the formation of helical filaments on single-stranded DNA and the subsequent search for homology in the duplex DNA, which results in formation of a synaptic intermediate consisting of three strands of DNA within a protein filament. Surprisingly, however, recent studies from several laboratories failed to detect helical filaments made by Dmc1 (28, This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA. Abbreviation: FRET, fluorescence resonance energy transfer.
|
|
|||||||||||||||
