Tetranucleotide Sequence Research Articles

Drosophila melanogaster is polymorphic for a specific repeated (CATA) sequence in the regulatory region of hsp23.

To identify sequence variation associated with a selection response for heat tolerance in Drosophila melanogaster, we sequenced 1400bp of the heat shock protein 23 gene (hsp23) promoter region in four heat-selected and two control lines. The region was found to be variable for a specific (CATA) repeated sequence, and the sequence CTT seems to be a hot spot for mutation. The repeated tetranucleotide sequence was located in several short repeats scattered throughout the entire region. Similar variable repeats are also located downstream the of hsp23 gene in the intergenic region between hsp23 and hsp27. We detected nine different hsp23 alleles. Their frequencies in the selection and control lines seemed to be mainly determined by genetic drift. The function of the CATA repeats is not yet known, though these regions have homology to SAR elements located in the intergenic region between two hsp70 genes, suggesting a similar function.

Inter-simple sequence repeat (ISSR) amplification for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza sativa L.)

Inter-simple sequence repeat (ISSR) amplification was used to analyze microsatellite motif frequency in the rice genome and to evaluate genetic diversity among rice cultivars. A total of 32 primers, containing different simple sequence repeat (SSR) motifs, were tested for amplification on a panel of 59 varieties, representative of the diversity of cultivated rice (Oryza sativa L.). The ISSR analysis provided insights into the organization, frequency and levels of polymorphism of different simple sequence repeats in rice. The more common dinucleotide motifs were more amenable to ISSR analysis than the more infrequent tri-, tetra- and penta-nucleotide motifs. The ISSR results suggested that within the dinucleotide class, the poly(GA) motif was more common than the poly(GT) motif and that the frequency and clustering of specific tri- and tetra-nucleotide simple sequence repeats was variable and motif-specific. Furthermore, trinucleotide ISSR markers were found to be less polymorphic than either dinucleotide or certain tetranucleotide ISSR markers, suggesting which motifs would be better targets for microsatellite marker development. The ISSR amplification pattern was used to group the rice genotypes by cluster analysis. These results were compared to surveys of the same varieties for amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP) and isozyme markers. The ISSR fingerprint could be used to differentiate the genotypes belonging to either Japonica or Indica sub species of cultivated rice and to dissect finer levels of diversity within each subspecies. A higher percentage of polymorphic bands was produced with the ISSR technique than the AFLP method, based on a similar PCR reaction. Therefore, ISSR amplification proved to be a valuable method for determining genetic variability among rice varieties and for rapidly identifying cultivars. This efficient genetic fingerprinting technique would be useful for characterizing the large numbers of rice accessions held in national and international germplasm centers.

Two Tetranucleotide Repeats within the Xq21.3/Yp11.2 Human Specific Region of Homology and Their Conservation in Primate Evolution

We locate two tetranucleotide repeat sequences (AT3 and T2C2) between the markers sY44 and sY46 within the Xq21.3/Yp block of homology that has been created since the separation of the chimpanzee and human lineages, and trace their origin in primate evolution. The T2C2 repeat is present only in hominoid primates. The sequence AT3 is present in Old World monkeys but not in New World monkeys, and has been lost in some gibbon species. In the bonobo, the AT3 repeat is the site of a new Alu insertion. These findings and their relationship to the conservation of other markers in this region cast light on the structure of a genomic region that has been subject to change in the course of primate evolution and may include one or more sites of instability.

Complexation of the antibiotic daunomycin with desoxytetraribonucleoside triphosphate 5’-d(CpGpCpG) in aqueous solution

This paper presents the results of our study of the complexation of the antibiotic daunomycin with desoxytetranucleotide 5’-d(CpGpCpG) in aqueous salt solution by one- and two-dimensional (2D-TOCSY and 2D-NOESY)1H NMR spectroscopy (500 MHz). The concentration and temperature dependences of the proton chemical shifts of molecules were measured and used to calculate the equilibrium reaction constants, the relative contents of different types of complex as functions of concentration and temperature, the limiting values of the proton chemical shifts of daunomycin in various complexes, and the thermodynamic parameters of complexation δH and δS. It is concluded that the triplet nucleotide sequences are the preferable sites at which daunomycin is attached. The binding of the second daunomycin molecule to both single-stranded and duplex forms of tetramer is markedly anticooperative. This is explained by the presence in the antibiotic of a positively charged amino sugar residue creating steric hindrances for the attachment of the second antibiotic molecule to the short tetranucleotide sequence. The most plausible spatial structure of the 1:2 complex of antibiotic with desoxytetranucleotide is constructed using the calculated values of the induced proton chemical shifts of daunomycin and 2D-NOE data.

The two homeodomains of the ZmHox2a gene from maize originated as an internal gene duplication and have evolved different target site specificities.

The maize ZmHox2a gene encodes two homeodomains which originated by a 699 bp duplication within an ancestral precursor. The sequences of the two ZmHox2a homeodomains are highly diverged in the N-terminal arm, while residues in the helical part have mostly been conserved. We show here that both ZmHox2a homeodomains are functional DNA-binding motifs but exhibit different target site specificities. CASTing experiments reveal a TCCT motif recognized by HD1 but a GATC tetranucleotide as the recognition sequence of HD2. Mutation of the central nucleotides in both tetranucleotide core motifs abolishes DNA binding. A domain swap experiment indicates that target site specificity is achieved in a combinatorial manner by the contributions of the diverged N-terminal arms together with the slightly different recognition helices. Computer modelling suggests that K47 and H54 in the recognition helices preferentially contact the bases at the 3'-terminus of the tetranucleotide target sequences.

Dissection of the sequence specificity of the Holliday junction endonuclease CCE1.

CCE1 is a Holliday (four-way DNA) junction-specific endonuclease which resolves mitochondrial DNA recombination intermediates in Saccharomycescerevisiae. The junction-resolving enzymes are a diverse class, widely distributed in nature from viruses to higher eukaryotes. In common with most other junction-resolving enzymes, the cleavage activity of CCE1 is nucleotide sequence-dependent. We have undertaken a systematic study of the sequence specificity of CCE1, using a single-turnover kinetic assay and a panel of synthetic four-way DNA junction substrates. A tetranucleotide consensus cleavage sequence 5'-ACT downward arrowA has been identified, with specificity residing mainly at the central CT dinucleotide. Equilibrium constants for CCE1 binding to four-way junctions are unaffected by sequence variations, suggesting that substrate discrimination occurs predominantly in the transition state complex. CCE1 cuts most efficiently at the junction center, but can also cleave the DNA backbone at positions one nucleotide 3' or 5' of the point of strand exchange, suggesting a significant degree of conformational flexibility in the CCE1:junction complex. Introduction of base analogues at single sites in four-way junctions has allowed investigation of the sequence specificity of CCE1 in finer detail. In particular, the N7 moiety of the guanine base-pairing with the cytosine of the consensus sequence appears to be crucial for catalysis. The functional significance of sequence specificity in junction-resolving enzymes is discussed.

Binding of two novel bisdaunorubicins to DNA studied by NMR spectroscopy.

In the search for new generations of anthracycline drugs, lower cytotoxic side effects and higher activity against resistant cancer cells are two major goals. A new class of bis-intercalating anthracycline drugs has been designed, synthesized, and shown to have promising activity against multidrug-resistant cells. Two daunorubicins symmetrically linked together via a p-xylenyl group, either at their N3' (compound WP631) or N4' sites (compound WP652), exhibit extraordinary DNA binding affinities. We have used high-resolution NRM studies to understand the DNA binding mode of these two new bis-daunorubicin anticancer compounds. The structures of the WP631-d(ACGTACGT)2 and the WP652-d(TGTACA)2 complexes have been determined by NOE-restrained refinement. WP631 binds strongly to the 5'-CG(A/T)(A/T)CG hexanucleotide sequence, with the aglycons intercalated between the two CpG sites at both ends of the hexanucleotide sequence. The overall conformation of the WP631-d(CGTACG)2 part is remarkably similar to the crystal structure of the 2:1 complex of daunorubicin and d(CGTACG)2, as predicted previously [Gao, Y.-G., & Wang, A.H.H. (1996), J. Biomol. Struct. Dyn. 13, 103-117]. In contrast, the related bis-intercalator WP652 prefers the 5'-PyGTPu tetranucleotide sequence, with the aglycons intercalated between the PypG and TpPu sites. The binding of WP652 to DNA results in a severely distroted B-DNA duplex with the p-xylenyl tether moiety significantly protruded away from the bottom of the minor groove. While WP652 in some ways behaves similarly to other anticancer bis-intercalating antibiotics (e.g., triostine A and echinomycin), the detailed interactions between those two classes of bis-intercalators are quite different.

In vitro genetic selection analysis of alfalfa mosaic virus coat protein binding to 3'-terminal AUGC repeats in the viral RNAs.

The coat proteins of alfalfa mosaic virus (AMV) and the related ilarviruses bind specifically to the 3' untranslated regions of the viral RNAs, which contain conserved repeats of the tetranucleotide sequence AUGC. The purpose of this study was to develop a more detailed understanding of RNA sequence and/or structural determinants required for coat protein binding by characterizing the role of the AUGC repeats. Starting with a complex pool of 39-nucleotide RNA molecules containing random substitutions in the AUGC repeats, in vitro genetic selection was used to identify RNAs that bound coat protein. After six iterative rounds of selection, amplification, and reselection, 25% of the RNAs selected from the randomized pool were wild type; that is, they contained all four AUGC sequences. Among the 31 clones analyzed, AUGC was clearly the preferred selected sequence at the four repeats, but some nucleotide sequence variability was observed at AUGC(865-868) if the other three AUGC repeats were present. Variant RNAs that bound coat protein with affinities equal to or greater than that of the wild-type molecule were not selected. To extend the in vitro selection results, RNAs containing specific nucleotide substitutions were transcribed in vitro and tested in coat protein and peptide binding assays. The data strongly suggest that the AUGC repeats provide sequence-specific determinants and contribute to a structural platform for specific coat protein binding. Coat protein may function in maintaining the 3' ends of the genomic RNAs during replication by stabilizing an RNA structure that defines the 3' terminus as the initiation site for minus-strand synthesis.

DNA repeats identify novel virulence genes in Haemophilus influenzae.

The whole genome sequence (1.83 Mbp) of Haemophilus influenzae strain Rd was searched to identify tandem oligonucleotide repeat sequences. Loss or gain of one or more nucleotide repeats through a recombination-independent slippage mechanism is known to mediate phase variation of surface molecules of pathogenic bacteria, including H. influenzae. This facilitates evasion of host defenses and adaptation to the varying microenvironments of the host. We reasoned that iterative nucleotides could identify novel genes relevant to microbe-host interactions. Our search of the Rd genome sequence identified 9 novel loci with multiple (range 6-36, mean 22) tandem tetranucleotide repeats. All were found to be located within putative open reading frames and included homologues of hemoglobin-binding proteins of Neisseria, a glycosyltransferase (lgtC gene product) of Neisseria, and an adhesin of Yersinia. These tetranucleotide repeat sequences were also shown to be present in two other epidemiologically different H. influenzae type b strains, although the number and distribution of repeats was different. Further characterization of the lgtC gene showed that it was involved in phenotypic switching of a lipopolysaccharide epitope and that this variable expression was associated with changes in the number of tetranucleotide repeats. Mutation of lgtC resulted in attenuated virulence of H. influenzae in an infant rat model of invasive infection. These data indicate the rapidity, economy, and completeness with which whole genome sequences can be used to investigate the biology of pathogenic bacteria.

Actinomycin D Binding to Single-stranded DNA: Sequence Specificity and Hemi-intercalation Model from Fluorescence and 1H NMR Spectroscopy

We have studied the sequence specificity in the binding of the potent antitumor drug actinomycin D (AMD) to single-stranded DNA (ssDNA) by fluorescence and NMR spectroscopy and by molecular modeling. The significant absorption and emission changes accompanying the interaction of the fluorescent derivative 7-amino-AMD with DNAs varying in length and base composition were used to calculate affinity constants for the drug – DNA complexes. The guanine-containing trinucleotide sequences AGT, AGA, and TGT embedded within 25-base oligonucleotides, constituted favorable binding sites. In contrast, the sequence TGA did not bind the drug appreciably. Among the DNAs studied, the highest affinity was for the tetranucleotide sequence TAGT. The binding was length dependent, an oligonucleotide of at least 14 bases being required for effective complex formation ( K a> 10 4M −1). AMD also bound to poly(d(AGT)). Gel electrophoresis confirmed that the complex was formed between the drug and individual unstructured DNA strands. The 1H NMR spectra of oligonucleotides containing the TAGT site and their complexes with AMD provided further insight into the mode(s) of interaction. A comparison of the measured chemical shifts with those estimated from ring-current calculations provided strong evidence for a hemi-intercalation of AMD between the A and G purine bases with a preference for one of two possible relative orientations. The latter were modeled as complexes with the sequence T 3AGT 3and refined by force field calculations with the AMBER program. The biological implications for this novel form of interaction of AMD with single-stranded DNA are discussed.

Formation of cyclobutane thymine dimers from UVA photosensitization of pyridopsoralen monoadducted DNA.

The present report provides evidence that thymine dimerization can be UVA photosensitized at a tetranucleotide, 5'-TATT-3', by a 7-methyl-pyrido(3,4-c)psoralen monoadduct in DNA. The efficiency of the photoprocess depends on the tetranucleotide flanking sequences. These results demonstrate that one DNA lesion can originate the contiguous formation of a second type of lesion and emphasize the sequence-specific response to interaction of drugs with DNA. Results are related to the sensitivity of DNA to 1,10-phenanthroline-cuprous ion complex nucleolytic activity and discussed in terms of the major role of local deformability of DNA in interaction with ligands.

Cloning and characterization of Sse9I DNA-methyltransferase recognizing 5'-AATT-3'.

The gene from Sporosarcina species 9D encoding Sse9I DNA-methyltransferase (M.Sse9I) was cloned and expressed in Escherichia coli. The recombinant plasmid pMSse-1 contains the M.Sse9I gene 1086 bp in length, corresponding to a protein of 362 amino acid residues. M.Sse9I recognizes the tetranucleotide sequence 5'-AATT-3' and modifies the second adenine within the recognition sequence. The amino acid sequence of M.Sse9I was compared with those of other methylases. According to mutual positions of four conservative domains the new enzyme belongs to a subgroup of D12 class. This subgroup includes Sse9I, CviAII, NlaIII and N-terminal domains of LlaI, FokI and StsI DNA-methyltransferases.

Genetic analysis of children of atomic bomb survivors.

Studies are under way for the detection of potential genetic effects of atomic bomb radiation at the DNA level in the children of survivors. In a pilot study, we have examined six minisatellites and five microsatellites in DNA derived from 100 families including 124 children. We detected a total of 28 mutations in three minisatellite loci. The mean mutation rates per locus per gamete in the six minisatellite loci were 1.5% for 65 exposed gametes for which mean parental gonadal dose was 1.9 Sv and 2.0% for 183 unexposed gametes. We detected four mutations in two tetranucleotide repeat sequences but no mutations in three trinucleotide repeat sequences. The mean mutation rate per locus per gamete was o% for the exposed gametes and 0.5% for the unexposed gametes in the five microsatellite loci. No significant differences in the mutation rates between the exposed and the unexposed gametes were detected in these repetitive sequences. Additional loci are being analyzed to increase the power of our study to observe a significant difference in the mutation rates at the 0.05 level of significance.

Linkage of congenital isolated adrenocorticotropic hormone deficiency to the corticotropin releasing hormone locus using simple sequence repeat polymorphisms

Genetic screening techniques using simple sequence repeat polymorphisms were applied to investigate the molecular nature of congenital isolated adrenocorticotropic hormone (ACTH) deficiency. We hypothesize that this rare cause of hypocortisolism shared by a brother and sister with two unaffected sibs and unaffected parents is inherited as an autosomal recessive single gene mutation. Genes involved in the hypothalamic-pituitary axis controlling cortisol sufficiency were investigated for a causal role in this disorder. Southern blotting showed no detectable mutations of the gene encoding pro-opiomelanocortin (POMC), the ACTH precursor. Other candidate genes subsequently considered were those encoding neuroendocrine convertase-1, and neuroendocrine convertase-2 (NEC-1, NEC-2), and corticotropin releasing hormone (CRH). Tests for linkage were performed using polymorphic di- and tetranucleotide simple sequence repeat markers flanking the reported map locations for POMC, NEC-1, NEC-2, and CRH. The chromosomal haplotypes determined by the markers flanking the loci for POMC, NEC-1, and NEC-2 were not compatible with linkage. However, 22 individual markers defining the chromosomal haplotypes flanking CRH were compatible with linkage of the disorder to the immediate area of this gene on chromosome 8. Based on these data, we hypothesize that the ACTH deficiency in this family is due to an abnormality of CRH gene structure or expression. These results illustrate the useful application of high density genetic maps constructed with simple sequence repeat markers for inclusion/exclusion studies of candidate genes in even very small nuclear families segregating for unusual phenotypes. © 1996 Wiley-Liss, Inc.

Linkage of congenital isolated adrenocorticotropic hormone deficiency to the corticotropin releasing hormone locus using simple sequence repeat polymorphisms.

Genetic screening techniques using simple sequence repeat polymorphisms were applied to investigate the molecular nature of congenital isolated adrenocorticotropic hormone (ACTH) deficiency. We hypothesize that this rare cause of hypocortisolism shared by a brother and sister with two unaffected sibs and unaffected parents is inherited as an autosomal recessive single gene mutation. Genes involved in the hypothalamic-pituitary axis controlling cortisol sufficiency were investigated for a causal role in this disorder. Southern blotting showed no detectable mutations of the gene encoding pro-opiomelanocortin (POMC), the ACTH precursor. Other candidate genes subsequently considered were those encoding neuroendocrine convertase-1, and neuroendocrine convertase-2 (NEC-1, NEC-2), and corticotropin releasing hormone (CRH). Tests for linkage were performed using polymorphic di- and tetranucleotide simple sequence repeat markers flanking the reported map locations for POMC, NEC-1, NEC-2, and CRH. The chromosomal haplotypes determined by the markers flanking the loci for POMC, NEC-1, and NEC-2 were not compatible with linkage. However, 22 individual markers defining the chromosomal haplotypes flanking CRH were compatible with linkage of the disorder to the immediate area of this gene on chromosome 8. Based on these data, we hypothesize that the ACTH deficiency in this family is due to an abnormality of CRH gene structure or expression. These results illustrate the useful application of high density genetic maps constructed with simple sequence repeat markers for inclusion/exclusion studies of candidate genes in even very small nuclear families segregating for unusual phenotypes.

The Efficiency of Translation Termination is Determined by a Synergistic Interplay Between Upstream and Downstream Sequences in Saccharomyces cerevisiae

In a recent study we found that the efficiency of translation termination could be decreased several hundred fold by altering the local sequence context surrounding stop codons in the yeast Saccharomyces cerevisiae. Suppression of termination was shown to be mediated by near-cognate tRNA mispairing with the termination codon. We have now examined in greater detail how the local sequence context affects the efficiency of translation termination in this organism. Our results indicate that the sequence immediately upstream of the termination codon plays a significant role in determining the efficiency of translation termination. An extended termination sequence (containing the stop codon and the following three nucleotides) was also found to be a major determinant of termination efficiency, with effects attributable to the fourth nucleotide being largely independent of the termination codon. For the UGA and UAA stop codons, the influence of the fourth position on termination efficiency (from most efficient to least efficient termination) was found to be G>U,A>C, while for the UAG codon it was U,A>C>G. These sequence-specific effects on the efficiency of translation termination suggest that polypeptide chain release factor (or another molecule that may play a role in translation termination, such as rRNA) recognizes an extended termination sequence in yeast. A previous study found a statistically significant bias toward certain tetranucleotide sequences (containing the stop codon and the first distal nucleotide) in several organisms. We found that tetranucleotide sequences most frequently used in yeast are among the most efficient at mediating translation termination, while rare tetranucleotide sequences mediate much less efficient termination. Taken together, our results indicate that upstream and downstream components of an extended sequence context act synergistically to determine the overall efficiency of translation termination in yeast.

Two thermostable type II restriction endonucleases from Icelandic strains of the genus Thermus: Tsp4C I (ACN/GT), a novel type II restriction endonuclease, and Tsp8E I, an isoschizomer of the mesophilic enzyme Bgl I (GCCNNNN/NGGC)

Sixteen isolates of thermophilic bacteria from the genus Thermus, isolated from neutral and alkaline hot water springs in the southwest region of Iceland, were tested for the presence of restriction endonucleases. Extracts from five of the isolates showed evidence of the presence of restriction endonuclease activity by producing discrete nucleotide fragments when incubated at 65 degrees C with lambda phage DNA. Two of the isolates (Tsp4C and Tsp8E) were found to have particularly high levels of restriction endonuclease activity, and the respective enzymes from these two Thermus isolates were partially purified and characterized and their recognition and cleavage sites were determined. Enzyme Tsp4C I is a novel Type II restriction endonuclease recognizing the interrupted palindromic tetranucleotide sequence ACNGT, where N can be any one of the four bases in DNA. Tsp4C I, which retains full enzyme activity when incubated for 10 min at temperatures up to 76 degrees C, hydrolyses the phosphodiester bond in both strands of a double-stranded DNA substrate between the third and fourth bases of the recognition sequence (ACN/GT), generating fragments with a single base 3'-OH overhang. Enzyme Tsp8E I is a thermostable isoschizomer of the mesophilic Type II restriction endonuclease Bgl I (GCCNNNN/NGGC) [Lee, Clanton and Chirikjiam (1979) Fed. Proc. 28, 294], generating fragments with a three base 3'-OH overhang. However, unlike Bgl I, Tsp8E I exhibits considerable thermal stability, retaining full enzyme activity when incubated for 10 min at temperatures up to 78 degrees C. Both Tsp4C I and Tsp8E I represent significant additions to the small but expanding list of the extremely thermostable restriction endonucleases.

Two mutant alleles of the insulin receptor gene in a family with a genetic form of insulin resistance: a 10 base pair deletion in exon 1 and a mutation substituting serine for asparagine-462.

Mutations in the insulin receptor gene cause several genetic syndromes associated with extreme insulin resistance. We have studied three insulin resistant siblings with acanthosis nigricans, dental abnormalities, and acral hypertrophy. The female patient also had primary amenorrhea due to hyperandrogenism. All three patients were compound heterozygotes with two mutant alleles of the insulin receptor gene. One allele had a 10-bp deletion in the region of exon 1 encoding the hydrophobic signal peptide; this leads to a frameshift and premature chain termination at codon 61. The deletion occurs at the site of a direct repeat of a hexanucleotide sequence interrupted by a tetranucleotide sequence; the deletion may have resulted from recombination between the upstream and downstream hexanucleotide repeats. In the other mutant allele, there is a missense mutation substituting serine for Asn462-a mutation identified previously in one allele of the insulin receptor gene in a patient with type-A insulin resistance. The Ser462 mutation impaired the ability of acidic pH to dissociate insulin from the receptor. Thus, Thus, like the previously described Glu460 mutation, the Ser462 mutation may retard dissociation of insulin from the receptor in the acidic compartment of the endosome and may, as a result, accelerate the rate of receptor degradation.

Salt dependence of calicheamicin-DNA site-specific interactions.

Calicheamicin gamma 1I site-specifically binds and cleaves three closely spaced tetranucleotide sequences embedded in an AT-rich region of a 142 base pair DNA restriction fragment. Cleavage is observed predominantly at the TCCT, TTGT, and ATCT sequences, of which TCCT is the primary cleavage site. The Gibbs free energies required to bind calicheamicin to these sequences within the DNA restriction fragment have been determined as a function of NaCl concentration at pH 8.1 and 0 degrees C and at pH 7.5 and 23 and 0 degrees C. Between 150 mM and 1 M NaCl, calicheamicin binding to all three sequences is insensitive to salt. The insensitivity of calicheamicin binding to salt continues to 50 mM NaCl for the TTGT and ATCT sequences; the delta G values for calicheamicin binding to these sequences are on the order of -7.8 to -7.9 kcal mol-1 over the entire range of NaCl concentrations studied. However, between 150 and 125 mM NaCl, the TCCT sequence displays a sharp transition in the delta G of calicheamicin binding from -7.6 to -8.9 kcal mol-1. Below 125 mM NaCl, the delta G values for calicheamicin binding to the TCCT sequence again are invariant. An analysis of the data in terms of polyelectrolyte theory suggests that counterion release from DNA does not contribute significantly to the energetics of the association and that the association of calicheamicin with specific DNA sequences is dominated by nonionic rather than electrostatic forces. Our results further suggest that some calicheamicin binding/cleavage sites are dependent on flanking sequences.

Genetic recombination in E. coli: RuvC protein cleaves Holliday junctions at resolution hotspots in vitro

The E. coli RuvC protein resolves Holliday junctions during genetic recombination and postreplication repair. Using recombination intermediates made by RecA protein, we have identified specific “hotspots” for RuvC resolution. Characterization of these sites reveals a common tetranucleotide sequence, with the consensus 5′- A T TT↓ G C -3′ . The correct orientation of the resolution site is required for cleavage. These observations suggest that the strand bias of this sequence will affect the outcome of recombinational crosses by directing resolution to either “patch” or “splice” recombinant products. Mutation of the consensus site in synthetic Holliday junctions abolishes or significantly reduces the efficiency of cleavage, although binding is unaffected, demonstrating that junction recognition and incision are biochemically separable events. We propose that efficient RuvC resolution requires the translocation of Holliday junctions to specific cleavage sites, thus providing a biochemical basis for the similar genetic defects observed in ruvA, ruvB, and ruvC mutants.