Meiotic recombination between homologous chromosomes ensures their proper segregation at the first division of meiosis and is the main force shaping genetic variation of genomes. The HOP2 and MND1 genes are essential for this recombination: Their disruption results in severe defects in homologous chromosome synapsis and an early-stage failure in meiotic recombination. The mouse Hop2 and Mnd1 proteins form a stable heterodimer (Hop2/Mnd1) that greatly enhances Dmc1-mediated strand invasion. In order to elucidate the mechanism by which Hop2/Mnd1 stimulates Dmc1, we identify several intermediate steps in the homologous pairing reaction promoted by Dmc1. We show that Hop2/Mnd1 greatly stimulates Dmc1 to promote synaptic complex formation on long duplex DNAs, a step previously revealed only for bacterial homologous recombinases. This synaptic alignment is a consequence of the ability of Hop2/Mnd1 to (1) stabilize Dmc1-single-stranded DNA (ssDNA) nucleoprotein complexes, and (2) facilitate the conjoining of DNA molecules through the capture of double-stranded DNA by the Dmc1-ssDNA nucleoprotein filament. To our knowledge, Hop2/Mnd1 is the first homologous recombinase accessory protein that acts on these two separate and critical steps in mammalian meiotic recombination.[Keywords: DNA repair; Dmc1 recombinase; homologous recombination; strand invasion; synaptic complex] Supplemental material is available at http://www.genesdev.org. Homologous recombination serves a critical function in the repair of DNA double-strand breaks (DSBs) and in the proper segregation of homologous chromosomes in meiosis (Kleckner 1996;Roeder 1997). Failure to establish a physical connection through chiasmata causes missegregation of chromosomes at prophase I and results in meiotic cell apoptosis or aneuploid gametes. The Dmc1 recombinase, a eukaryotic homolog of the bacterial RecA protein, is expressed exclusively in meiotic cells and is a major player in meiotic homologous recombination. It promotes the search for homology and catalyzes the invasion of a single-stranded end generated by the 5Ј resection of DSBs introduced by Spo11 into a homologous unbroken double-stranded DNA (dsDNA) to form joint molecules through strand invasion (D-loop formation) (Li et al. 1997;Masson et al. 1999;Hong et al. 2001;Masson and West 2001;Neale and Keeney 2006). The interaction between Hop2, Mnd1, and Dmc1 and/or Rad51, the ubiquitously expressed eukaryotic homolog of RecA, is crucial for the progression of meiotic homologous recombination. Biochemical studies have shown that the Hop2/Mnd1 complex physically interacts with and stimulates Dmc1 and Rad51 strand invasion activity (Chen et al. 2004;Petukhova et al. 2005;Pezza et al. 2006). The cooperation between Dmc1/Rad51 and Hop2/ Mnd1 is likely to be crucial in vivo, since without Hop2 and/or Mnd1, in yeast (Leu et al. 1998;Gerton and DeRisi 2002; Roeder 2002, 2003;Zierhut et al. 2004;Henry et al. 2006), Arabidopsis thaliana (Domenichini et al. 2006;Kerzendorfer et al. 2006;Panoli et al. 2006), and mouse (Pe...

We have recently shown that under superhelical stress and/or acid pH the homopurine-homopyrimidine tracts conforming to the mirror symmetry (H palindromes) form a novel DNA structure, the H form. According to our model, the H form includes (1) a triplex formed by half of the purine strand and by the homopyrimidine hairpin and (2) the unstructured other half of the purine strand. We used four specially designed sequences to demonstrate that, in accordance with our model, the mirror symmetry is essential for facile formation of the H form detected by two-dimensional gel electrophoresis. Here we report that, under conditions favouring the H-form extrusion, guanines of the 3' half of the purine strand are protected against alkylation by dimethylsulphate, whereas adenines of the 5' half of the purine strand react with diethyl pyrocarbonate. These data indicate that the 3' half of the homopurine strand is within the triplex whereas the 5' half is unstructured, in full agreement with our model.

dinG was identified as a DNA damage-inducible gene in a genetic screen scoring for induction of the transcription of galactokinase gene fusions after treatment of Escherichia coli cells with mitomycin C. Transcription of the dinG::galK fusion was suppressed by overexpression of the LexA protein, suggesting the SOS nature of the induction (1). Indeed, sequencing of the dinG promoter revealed an asymmetric nucleotide sequence TTG(N 10 )CAG that was similar but not identical to the canonical, fully symmetrical CTG(N 10 )CAG SOS box. Despite the deviation of the SOS box found in the dinG regulatory region from the consensus sequence of the LexA-binding box, the double-stranded (ds) 1 oligonucleotide TTGG(N 8 )ACAG bound the LexA repressor with high affinity in an electrophoretic mobility shift assay (2). The dinG promoter was also up-regulated upon DNA damage by nalidixic acid (3).dinG, along with lexA and dinI, was isolated in another genetic screen aimed at isolating multicopy suppressors of the cold-sensitive phenotype of the DinD68 mutation. This particular mutation in the DNA damage-inducible dinD gene, which is also regulated by the LexA-RecA system, results in the constitutive expression of the SOS response at lowered temperature (Ͻ20°C) (4). Because both dinG and dinI are part of the SOS response (1, 2, 5) and they suppress an SOS phenotype of the dinD68 mutation (6), dinG could also be a negative regulator of the SOS response in a manner similar to dinI (7,8).Analysis of the protein sequence of E. coli dinG reveals that it encodes a putative DNA helicase related to yeast DNA helicases Chl1 and Rad3 from Saccharomyces cerevisiae, Rad15 from Schizosaccharomyces pombe, and the human helicases XPD and BACH1 (9, 10). The mutant forms of the last two proteins result in well described human diseases, three human recessive photosensitive syndromes for XPD, and early onset breast cancer for BACH1 (10, 11). DinG and its eukaryotic counterparts have been classified as superfamily II helicases on the basis of the presence of seven canonical helicase motifs in their sequences (9,12,13). Still, the presence of the helicasespecific motifs in the protein amino acid sequence per se does not necessarily imply that it is a bona fide helicase. Proteins having helicase motifs but lacking a helicase activity are well known. Among them are the endonuclease (R) subunits of type I and type III restriction-modification enzymes (14), both bacterial and human transcription-repair coupling factors Mfd (15), and CSB/ERCC6 (16), members of SWI2/SNF2 family chromatin remodeling factors (17) and the RAD54 recombinational DNA repair protein (18).To prove that DinG is a true helicase, we carried out the purification and biochemical characterization of the E. coli DinG protein. In agreement with the prediction (9), DinG possesses DNA-dependent ATPase and helicase activities. We discuss the possible biological role that DinG helicase might play. EXPERIMENTAL PROCEDURESBacterial Strains and Plasmids-Gene deletions were created using a combinati...

When DinI is present at concentrations that are stoichiometric with those of RecA or somewhat greater, DinI has a substantial stabilizing effect on RecA filaments bound to DNA. Exchange of RecA between free and bound forms was almost entirely suppressed, and highly stable filaments were documented with several different experimental methods. DinI-mediated stabilization did not affect RecA-mediated ATP hydrolysis and LexA co-protease activities. Initiation of DNA strand exchange was affected in a DNA structure-dependent manner, whereas ongoing strand exchange was not affected. Destabilization of RecA filaments occurred as reported in earlier work but only when DinI protein was present at very high concentrations, generally superstoichiometric, relative to the RecA protein concentration. DinI did not facilitate RecA filament formation but stabilized the filaments only after they were formed. The interaction between the RecA protein and DinI was modulated by the C terminus of RecA. We discuss these results in the context of a new hypothesis for the role of DinI in the regulation of recombination and the SOS response.The RecA protein plays a principle role in the processes of homologous recombinational DNA repair (reviewed in Refs.