Activation of Human Meiosis-specific Recombinase Dmc1 by Ca2+

Abstract

Rad51 and its meiotic homologIn most eukaryotic organisms, meiotic recombination is required for generation of genetic diversity and for accurate segregation of homologous chromosomes (1-3). Mutations that impair meiotic recombination have been reported to increase non-disjunction in Schizosaccharomyces pombe, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila, mice, and humans; malfunction of meiotic recombination is a leading cause of trisomies in humans (4).The molecular mechanisms of meiotic recombination have been extensively studied, especially in yeast. Meiotic recombination is initiated by DNA double strand breaks introduced in specific regions of chromosomes by a specialized enzyme, Spo11 (5, 6). Consequently, the mechanism of meiotic recombination appears to be closely related to the process of double strand break repair (7-10). The double-stranded DNA (dsDNA) 1 ends are resected to produce an intermediate with a 3Ј-overhanging single-stranded DNA (ssDNA) tail in a process that involves the Mre11/Rad50/Xrs2 heterotrimer (11). The consequent steps of meiotic recombination require Rad51 and Dmc1 proteins and other members of the Rad52 epistasis group (12). It is known that both Rad51 and Dmc1 can polymerize on the ssDNA tails to form extended nucleoprotein complexes that promote a search for homologous DNA and that perform DNA strand exchange (13-17). Rad51 and Dmc1 proteins have significant structural homology, sharing 52% identical amino acid residues, and display similar biochemical properties. However, these proteins may play distinct roles in vivo. Although Rad51 protein is expressed in both mitotic and meiotic cells, Dmc1 is a meiosis-specific protein. In mammals, Rad51 is an essential protein; RAD51 Ϫ/Ϫ knockouts in the mouse cause embryonic lethality. In contrast, DMC1 Ϫ/Ϫ knockouts are viable, but sterile (18,19).In vitro, under standard conditions in the presence of ATP and Mg 2ϩ , human (h) Dmc1 protein forms a peculiar structure on ssDNA defined as stacks of octameric rings (20) and weakly promotes DNA strand exchange (15, 21). Recently, it was found that KCl at elevated concentrations (100 -200 mM) strongly stimulates the DNA strand exchange activity of hDmc1 protein and also promotes the formation of hDmc1⅐ssDNA helical nucleoprotein filaments (17).Previously, we demonstrated that Ca 2ϩ activates the DNA strand exchange activity of hRad51 protein (22) and that Ca 2ϩ is required for stimulation of hRad51 protein by hRad54 protein (23). Here, we examine the effect of Ca 2ϩ on the DNA strand exchange activity of hDmc1. The results show that the DNA strand exchange activity of hDmc1 protein is greatly enhanced by Ca 2ϩ . Taken together with our previous findings, the results suggest that Ca 2ϩ may play a role in regulation of homologous recombination in vivo. This role of Ca 2ϩ seems especially compelling because (i) Ca 2ϩ is known as a common intracellular regulator (24), and (ii) Ca 2ϩ concentration rises in response to DNA damage (25-30) and during meiosis (31), at the time when ho...

Clarifying the mechanics of DNA strand exchange in meiotic recombination

“…More recently, a robust Dmc1-mediated strand exchange reaction has been described, which differs from previous studies in using physiological pH and higher salt concentrations 26,[34][35][36] . Under these conditions, helical Dmc1-ssDNA filaments were formed 26,35,37 (Fig.…”

“…Ca 2+ stimulates both filament formation on ssDNA and DNA strand exchange by bacterially expressed human and yeast Dmc1 proteins 35,36 . Ca 2+ also stimulates Rad51 (ref 39), so it has been suggested that Dmc1 and Rad51 activity might be modulated in vivo by variation in intracellular calcium levels 35,36 .…”

Saccharomyces cerevisiae Dmc1 and Rad51 Proteins Preferentially Function with Tid1 and Rad54 Proteins, Respectively, to Promote DNA Strand Invasion during Genetic Recombination

“…In the case of RAD51, Ca 2ϩ maintains an active presynaptic filament by blocking ATP hydrolysis and thereby preventing the self-inhibition that accompanies accumulation of ADP within the filament. In contrast, for DMC1, Ca 2ϩ does block ATP hydrolysis, but this is not the basis for stimulation because a similar stimulation by Ca 2ϩ is observed in the presence of the non-hydrolyzable AMP-PNP; thus, Ca 2ϩ apparently induces a conformational change in the filament assembled on ssDNA that enables more effective DNA strand exchange (35,42). For yeast Dmc1, the modulation by Ca 2ϩ is similar to human DMC1 in that Ca 2ϩ also stimulates joint molecule formation slightly when AMP-PNP is used (supplemental Fig.…”

“…1C) established that the protein binds ssDNA. Previous reports established that yeast, as well as human, Dmc1 forms stable filaments only in the presence of calcium (34,35). We therefore examined the ability of Dmc1 to form complexes with ssDNA in the presence of magnesium acetate or calcium acetate (henceforth referred to as Mg 2ϩ and Ca 2ϩ , respectively).…”

Red-Hed regulation: recombinase Rad51, though capable of playing the leading role, may be relegated to supporting Dmc1 in budding yeast meiosis: Figure 1.

“…However, subsequent studies showed that, under certain conditions, Dmc1 forms right-handed filaments similar to those formed by the other recombinases (Sehorn et al 2004;Burgreev et al 2005;Lee et al 2005). Interestingly, substitution of Mg 2+ with Ca 2+ resulted in formation of long helical filaments on single-stranded DNA (ssDNA); Ca 2+ stimulated Dmc1's strand assimilation and strand exchange activity as well (Bugreev et al 2005;Lee et al 2005). Although it remains to be seen if Ca 2+ is a biologically relevant modifier of Dmc1 activity, it is clear that conditions favoring filament formation enhance recombinase activity.…”