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.

The Escherichia coli DinG protein is a DNA damage-inducible member of the helicase superfamily 2. Using a panel of synthetic substrates, we have systematically investigated structural requirements for DNA unwinding by DinG. We have found that the helicase does not unwind blunt-ended DNAs or substrates with 3-ss tails. On the other hand, the 5-ss tails of 11-15 nucleotides are sufficient to initiate DNA duplex unwinding; bifurcated substrates further facilitate helicase activity. DinG is active on 5-flap structures; however, it is unable to unwind 3-flaps. Similarly to the homologous Saccharomyces cerevisiae Rad3 helicase, DinG unwinds DNA⅐RNA duplexes. DinG is active on synthetic D-loops and R-loops. The ability of the enzyme to unwind D-loops formed on superhelical plasmid DNA by the E. coli recombinase RecA suggests that D-loops may be natural substrates for DinG. Although the availability of 5-ssDNA tails is a strict requirement for duplex unwinding by DinG, the unwinding of D-loops can be initiated on substrates without any ss tails. Since DinG is DNA damage-inducible and is active on D-loops and forked structures, which mimic intermediates of homologous recombination and replication, we conclude that this helicase may be involved in recombinational DNA repair and the resumption of replication after DNA damage.dinG is a damage-inducible (1-5) SOS-regulated (1) gene encoding for a superfamily 2 DNA helicase (6, 7) related to DNA helicases Chl1 and Rad3 from Saccharomyces cerevisiae, Rad15 from Schizosaccharomyces pombe, and the human helicases XPD and BACH1 (6, 8 -11). XPD, a 5Ј 3 3Ј helicase, is a part of the multisubunit complex TFIIH that plays a dual role in the initiation of transcription from RNA polymerase II promoters and in nucleotide excision repair (NER) 2 (12). TFIIH participates in both subpathways of NER: global genome NER and transcription-coupled NER (13). Mutations interfering with the proper function of XPD helicase in humans result in three rare recessive photosensitive syndromes: xeroderma pigmentosum, xeroderma pigmentosum/Cockayne syndrome, and tricothiodystrophy (13). Mutations in BACH1, the protein interacting with the breast cancer susceptibility factor BRCA1, cause an early onset familiar breast cancer (9); one of those mutations was shown to abrogate the ATPase and helicase activity of BACH1 in vitro (8). BACH1, also known as BRIP1, was recently identified as the gene defective in the J complementation group of Fanconi anemia, FANCJ, (14).Although the functions of XPD and its complexes are intensely studied and fairly well understood, much more has to be learned about BACH1/BRP1/FANCJ and its role in the Fanconi anemia pathway and in cancer susceptibility. As related helicases, BACH1 and DinG proteins could be potentially involved in similar aspects of nucleic acids metabolism. Thus, genetic, mechanistic, and structural studies of the quite tractable bacterial DinG protein could shed some light on the function of human BACH1.Recently, we purified the Escherichia coli DinG protein an...

A differential effect is found of various bivalent cations (Ba2+, Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+, Zn2+ and Hg2+) on stability of intermolecular Py-Pu-Pu triplex with different sequence of base triads. Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ do stabilize the d(C)n d(G)n d(G)n triplex whereas Ba2+ and Hg2+ do not. Ba2+, Ca2+, Mg2+ and Hg2+ destabilize the d(TC)n d(GA)n d(AG)n triplex whereas Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ stabilize it. The complexes we observe are rather stable because they do not dissociate during time of gel electrophoresis in the co-migration experiments. Chemical probing experiments with dimethyl sulfate as a probe indicate that an arbitrary homopurine-homopyrimidine sequence forms triplex with corresponding purine oligonucleotide in the presence of Mn2+ or Zn2+, but not Mg2+. In the complex the purine oligonucleotide has antiparallel orientation with respect to the purine strand of the duplex. Specifically, we have shown the formation of the Py-Pu-Pu triplex in a fragment of human papilloma virus HPV-16 in the presence of Mn2+.

The molecular structure of the Escherichia coli RecA protein in the absence of DNA revealed two disordered or mobile loops that were proposed to be DNA binding sites. A short peptide spanning one of these loops was shown to carry out the key reaction mediated by the whole RecA protein: pairing (targeting) of a single-stranded DNA to its homologous site on a duplex DNA. In the course of the reaction the peptide bound to both substrate DNAs, unstacked the single-stranded DNA, and assumed a beta structure. These events probably recapitulate the underlying molecular pathway or mechanism used by homologous recombination proteins.

FANCJ mutations are genetically linked to the Fanconi anemia complementation group J and predispose individuals to breast cancer. Understanding the role of FANCJ in DNA metabolism and how FANCJ dysfunction leads to tumorigenesis requires mechanistic studies of FANCJ helicase and its protein partners. In this work, we have examined the ability of FANCJ to unwind DNA molecules with specific base damage that can be mutagenic or lethal. FANCJ was inhibited by a single thymine glycol, but not 8-oxoguanine, in either the translocating or nontranslocating strands of the helicase substrate. In contrast, the human RecQ helicases (BLM, RECQ1, and WRN) display strand-specific inhibition of unwinding by the thymine glycol damage, whereas other DNA helicases (DinG, DnaB, and UvrD) are not significantly inhibited by thymine glycol in either strand. In the presence of replication protein A (RPA), but not Escherichia coli single-stranded DNA-binding protein, FANCJ efficiently unwound the DNA substrate harboring the thymine glycol damage in the nontranslocating strand; however, inhibition of FANCJ helicase activity by the translocating strand thymine glycol was not relieved. Strand-specific stimulation of human RECQ1 helicase activity was also observed, and RPA bound with high affinity to single-stranded DNA containing a single thymine glycol. Based on the biochemical studies, we propose a model for the specific functional interaction between RPA and FANCJ on the thymine glycol substrates. These studies are relevant to the roles of RPA, FANCJ, and other DNA helicases in the metabolism of damaged DNA that can interfere with basic cellular processes of DNA metabolism. Fanconi anemia (FA)2 is an autosomal recessive disorder characterized by multiple congenital anomalies, progressive bone marrow failure, and high cancer risk (1-3). Cells from FA patients exhibit spontaneous chromosomal instability and hypersensitivity to agents that induce DNA interstrand crosslinks. Although the precise mechanistic details of the FA pathway of interstrand cross-link-repair are not well understood, progress has been made in the identification of the FA proteins that are required for the pathway (1-3). Among the 13 FA complementation groups from which all FA genes have been cloned, only a few of the FA proteins are predicted to have direct roles in DNA metabolism. One of the more recently identified FA proteins shown to be responsible for complementation of the FA complementation group J is FANCJ (4 -6). FANCJ was originally designated BACH1 (BRCA1-associated C-terminal helicase), which was discovered by Cantor et al. (7) as a protein that binds to the BRCT repeats of BRCA1. A genetic interaction between FANCJ and BRCA1 in double strand break repair was established (7), and FANCJ mutations were identified in early onset breast cancer (7-9), suggesting a tumor suppressor role of FANCJ.FANCJ was first shown to be a DNA-dependent ATPase that catalytically unwinds duplex DNA with a 5Ј to 3Ј directionality (10). Consistent with its directionality, FANCJ r...