DM2 CCTG*CAGG repeats are crossover hotspots that are more prone to expansions than the DM1 CTG*CAG repeats in Escherichia coli

Myotonic dystrophy type 2 (DM2) is caused by the extreme expansion of the repeating tetranucleotide CCTG*CAGG sequence from <30 repeats in normal individuals to approximately 11,000 for the full mutation in certain patients. This repeat is in intron 1 of the zinc finger protein 9 gene on chromosome 3q21. Since prior work demonstrated that CTG*CAG and GAA*TTC triplet repeats (responsible for DM1 and Friedreich's ataxia, respectively) can expand by genetic recombination, we investigated the capacity of the DM2 tetranucleotide repeats to also expand during this process. Both gene conversion and unequal crossing over are attractive mechanisms to effect these very large expansions. (CCTG*CAGG)n (where n=30, 75, 114 or 160) repeats showed high recombination crossover frequencies (up to 27-fold higher than the non-repeating control) in an intramolecular plasmid system in Escherichia coli. Furthermore, a distinct orientation effect was observed where orientation II (CAGG on the leading strand template) was more prone to recombine. Expansions of up to double the length of the tetranucleotide repeats were found. Also, the repeating tetranucleotide sequence was more prone to expansions (to give lengths longer than a single repeating tract) than deletions as observed for the CTG*CAG and GAA*TTC repeats. We determined that the DM2 tetranucleotide repeats showed a lower thermodynamic stability when compared to the DM1 trinucleotide repeats, which could make them better targets for DNA repair events, thus explaining their expansion-prone behavior. Genetic studies in SOS-repair mutants revealed high frequencies of recombination crossovers although the SOS-response itself was not induced. Thus, the genetic instabilities of the CCTG*CAGG repeats may be mediated by a recombination-repair mechanism that is influenced by DNA structure.     

Analysis of Drosophila melanogaster satellite IV with restriction endonuclease MboII.

The products of digestion of Drosophila melanogaster satellite IV DNA with restriction endonuclease MboII have been analysed and found to be consistent with a repeating pentamer sequence (A-G-A-A-G)_n for satellite IV. More than 95% of the satellite DNA is digested to fragments less than 25 base-pairs in length, suggesting that the DNA sequence is highly conserved.     

DnaB helicase affects the initiation specificity of Escherichia coli primase on single-stranded DNA templates

The effect of DnaB helicase on the initiation specificity of primase was studied biochemically using a series of single-stranded DNA templates in which each nucleotide of the trinucleotide d(CTG) initiation sequence was systematically varied. DnaB helicase accelerated the rate of primer syntheisis, prevented "overlong" primers from forming and decreased the initiation specificity of primase. In the presence of DnaB helicase, all trinucleotides could serve as the primer initiation site although there was a distinct preference for d(CAG). These data may explain the high chromosomal prevalence of octanucleotides containing CTG on the leading strand and its complement CAG on the lagging strand. The specificity of DnaB helicase places it on the lagging strand template where it stimulates the initiation of Okazaki fragment synthesis. In the absence of DnaB helicase, primase preferentially primed the d(CTG) template. In the presence of DnaB helicase, the initiation preference was not only altered but also the preferred initiating nucleotide was found to be GTP rather than ATP, for both the d(CTG) and the d(CAG) templates. This suggested that the specificity of primase for the d(CTG) initiation trinucleotide was predominantly unaffected in the absence of DnaB helicase on short ssDNA templates, whereas in conjunction with DnaB helicase, the specificity was altered and this alteration has significant implications in the replication of Escherichia coli chromosome in vivo.     

Long CTG.CAG repeat sequences markedly stimulate intramolecular recombination

Previous studies have shown that homologous recombination is a powerful mechanism for generation of massive instabilities of the myotonic dystrophy CTG.CAG sequences. However, the frequency of recombination between the CTG.CAG tracts has not been studied. Here we performed a systematic study on the frequency of recombination between these sequences using a genetic assay based on an intramolecular plasmid system in Escherichia coli. The rate of intramolecular recombination between long CTG.CAG tracts oriented as direct repeats was extraordinarily high; recombinants were found with a frequency exceeding 12%. Recombination occurred in both RecA(+) and RecA(-) cells but was approximately 2-11 times higher in the recombination proficient strain. Long CTG.CAG tracts recombined approximately 10 times more efficiently than non-repeating control sequences of similar length. The recombination frequency was 60-fold higher for a pair of (CTG.CAG)(165) tracts compared with a pair of (CTG.CAG)(17) sequences. The CTG.CAG sequences in orientation II (CTG repeats present on a lagging strand template) recombine approximately 2-4 times more efficiently than tracts of identical length in the opposite orientation relative to the origin of replication. This orientation effect implies the involvement of DNA replication in the intramolecular recombination between CTG.CAG sequences. Thus, long CTG.CAG tracts are hot spots for genetic recombination.     

Orientation dependence of trinucleotide CAG repeat instability in Saccharomyces cerevisiae.

To examine the chromosomal stability of repetitions of the trinucleotide CAG, we have cloned CAG repeat tracts onto the 3' end of the Saccharomyces cerevisiae ADE2 gene and placed the appended gene into the ARO2 locus of chromosome VII. Examination of chromosomal DNA from sibling colonies arising from clonal expansion of strains harboring repeat tracts showed that repeat tracts often change in length. Most changes in tract length are decreases, but rare increases also occur. Longer tracts are more unstable than smaller tracts. The most unstable tracts, of 80 to 90 repeats, undergo changes at rates as high as 3 x 10(-2) changes per cell per generation. To examine whether repeat orientation or adjacent sequences alter repeat stability, we constructed strains with repeat tracts in both orientations, either with or without sequences 5' to ADE2 harboring an autonomously replicating sequence (ARS; replication origin). When CAG is in the ADE2 coding strand of strains harboring the ARS, the repeat tract is relatively stable regardless of the orientation of ADE2. When CTG is in the ADE2 coding strand of strains harboring the ARS, the repeat tract is relatively unstable regardless of the orientation of ADE2. Removal of the ARS as well as other sequences adjacent to the 5' end of ADE2 alters the orientation dependence such that stability now depends on the orientation of ADE2 in the chromosome. These results suggest that the proximity of an ARS or another sequence has a profound effect on repeat stability.     

Cleavage of single strand oligonucleotides and bacteriophage phi X174 DNA by Msp I endonuclease

Type II restriction endonucleases cleave duplex DNA at nucleotide sequences displaying 2-fold symmetry. Our data show that Msp I cleaves single strand oligonucleotides, d(G-A-A-C-C-G-G-A-G-A) and d(T-C-T-C-C-G-G-T-T) at 4 degrees, 25 degrees, and 37 degrees C reaction temperatures. The rate of cleavage of d(G-A-A-C-C-G-G-A-G-A) is several-fold faster than that of d(T-C-T-C-C-G-G-T-T). Single strand phi X174 DNA is also, cleaved by Msp I endonuclease giving well defined fragments. 5'-Nucleotide analysis of the fragments generated from single strand and replicating form DNA suggest that cleavage occurs at the recognition sequence d(C-C-G-G). The data show that Msp I endonuclease cleaves single strand oligonucleotides and prefers a recognition sequence surrounded by purine nucleotides. A general model for endonuclease cleavage of single strand and duplex DNA is presented.     

Weak strand displacement activity enables human DNA polymerase beta to expand CAG/CTG triplet repeats at strand breaks

Using synthetic DNA constructs in vitro, we find that human DNA polymerase beta effectively catalyzes CAG/CTG triplet repeat expansions by slippage initiated at nicks or 1-base gaps within short (14 triplet) repeat tracts in DNA duplexes under physiological conditions. In the same constructs, Escherichia coli DNA polymerase I Klenow Fragment exo(-) is much less effective in expanding repeats, because its much stronger strand displacement activity inhibits slippage by enabling rapid extension through two downstream repeats into flanking non-repeat sequence. Polymerase beta expansions of CAG/CTG repeats, observed over a 32-min period at rates of approximately 1 triplet added per min, reveal significant effects of break type (nick versus gap), strand composition (CTG versus CAG), and dNTP substrate concentration, on repeat expansions at strand breaks. At physiological substrate concentrations (1-10 microm of each dNTP), polymerase beta expands triplet repeats with the help of weak strand displacement limited to the two downstream triplet repeats in our constructs. Such weak strand displacement activity in DNA repair at strand breaks may enable short tracts of repeats to be converted into longer, increasingly mutable ones associated with neurological diseases.     

Selective cleavage and anticoagulant activity of a sulfated fucan: stereospecific removal of a 2-sulfate ester from the polysaccharide by mild acid hydrolysis, preparation of oligosaccharides, and heparin cofactor II-dependent anticoagulant activity

A linear sulfated fucan with a regular repeating sequence of [3)-alpha-L-Fucp-(2SO4)-(1-->3)-alpha-L-Fucp-(4SO4)-(1-->3)-alpha-L-Fucp-(2,4SO4)-(1-->3)-alpha-L-Fucp-(2SO4)-(1-->]n is an anticoagulant polysaccharide mainly due to thrombin inhibition mediated by heparin cofactor II. No specific enzymatic or chemical method is available for the preparation of tailored oligosaccharides from sulfated fucans. We employ an apparently nonspecific approach to cleave this polysaccharide based on mild hydrolysis with acid. Surprisingly, the linear sulfated fucan was cleaved by mild acid hydrolysis on an ordered sequence. Initially a 2-sulfate ester of the first fucose unit is selectively removed. Thereafter the glycosidic linkage between the nonsulfated fucose residue and the subsequent 4-sulfated residue is preferentially cleaved by acid hydrolysis, forming oligosaccharides with well-defined size. The low-molecular-weight derivatives obtained from the sulfated fucan were employed to determine the requirement for interaction of this polysaccharide with heparin cofactor II and to achieve complete thrombin inhibition. The linear sulfated fucan requires significantly longer chains than mammalian glycosaminoglycans to achieve anticoagulant activity. A slight decrease in the molecular size of the sulfated fucan dramatically reduces its effect on thrombin inactivation mediated by heparin cofactor II. Sulfated fucan with approximately 45 tetrasaccharide repeating units binds to heparin cofactor II but is unable to link efficiently the plasma inhibitor and thrombin. This last effect requires chains with approximately 100 or more tetrasaccharide repeating units. We speculate that the template mechanism may predominate over the allosteric effect in the case of the linear sulfated fucan inactivation of thrombin in the presence of heparin cofactor II.     

Sites of preferential induction of cyclobutane pyrimidine dimers in the nontranscribed strand of lacI correspond with sites of UV-induced mutation in Escherichia coli

An approach utilizing fluorescence-activated DNA sequencing technology was used to study the position and frequency of UV-induced lesions in the lacI gene of Escherichia coli. The spectrum of sites of UV damage in the NC+ region of the gene was compared with a published spectrum of UV-induced mutation in lacI (Schaaper, R.M., Dunn, R.L., and Glickman, B.W. (1987) J. Mol. Biol. 198, 187-202). On average, the frequency of UV-induced lesions in the nontranscribed strand was higher than that in the transcribed strand in the region analyzed. A large fraction of mutations occurs at sites of UV-induced lesions in the nontranscribed strand, but not in the transcribed strand. This bias is reduced in an excision repair deficient (UvrB-) strain. In addition, mutations occur overwhelmingly at sites where a dipyrimidine sequence is present in the nontranscribed strand. This bias is also markedly reduced in the UvrB- strain. In light of recent work Mellon and Hanawalt (Mellon, I., and Hanawalt, P.C. (1989) Nature 342, 95-98) describing the preferential removal of cyclobutane dimers from the transcribed strand of the expressed lacZ gene in E. coli, our data suggest that preferential strand repair may have a significant effect on mutagenesis.     

Isolation of a short, cytosine-rich repeating unit from the DNA of Escherichia coli

Hybridization of heterologous nucleic acids has provided the means for isolating a repeating sequence which is located next to template regions of DNA. Separated single strands of 32P-labelled DNA from Escherichia coli were to a limited extent able to anneal with DNA of Micrococcus lysodeikticus immobilized on nitrocellulose membrane filters. The resulting hybrid was resistant to enzymes specific for unpaired strands, nuclease S1 (Aspergillus oryzae) and exonuclease I (E. coli). The E. coli DNA so hybridized was isolated and characterized. It contained all four bases with cytosine predominating; strand length was about 50-60 nucleotides. Since these units occupied about 1-2% of the length of the E. coli chromosome, they would have to be repeated about 2000 times in a single cell. Formation of the unusual hybrid was not diminished by prior saturation of the E. coli DNA with homologous 3H-labelled RNA. In fact both RNA and additional increments of DNA were detected on the filters approximately in a 1:1 ratio, showing that some of the repeating sequences were physically continuous with transcribed regions of DNA.     

Amino acid sequence of a segment of the Acanthamoeba myosin II heavy chain containing all three regulatory phosphorylation sites

The actin-activated Mg2+-ATPase activity of myosin II from the soil amoeba Acanthamoeba castellanii is regulated by phosphorylation of 3 serine residues on the myosin II heavy chain. Partial chymotryptic digestion of 32P-labeled myosin II cleaves from the tail end of the myosin II heavy chain a small peptide which contains all three phosphorylation sites. During purification the phosphorylated peptide is resolved into several different species as a result of heterogeneity both in phosphate content and in size (probably due to chymotryptic cleavage at the carboxyl terminus). However, all forms of the peptide have an identical amino terminus. The sequence of the first 58 residues of the peptide is: N-S-A-L-E-S-D-K-Q-I10-L-E-D-E-I-G-D-L-H- E20-K-N-K-Q-L-Q-A-K-I-A30-Q-L-Q-D-E-I-D-G-T- P40-S-S-R-G-G-S-T-R-G-A50-S-A-R-G-A-S-V-R. The phosphorylated serines are at positions 46, 51, and 56. The first 36 residues of the sequence display a repeating 3-4-3-4 pattern of hydrophobic residues suggesting that this section of the peptide forms an alpha-helical coiled-coil structure. A -Gly-Thr-Pro sequence at residues 38-40 disrupts the alpha-helix and, at the same point, the repeating pattern of non-polar residues is lost. It is likely that the residues extending from Gly-38 to the end of the myosin II tail, which include the 3 phosphorylatable serines, form a randomly coiled or small globular structure. This is the first report of the sequence around the regulatory phosphorylation sites on any myosin heavy chain.     

DNA double-strand breaks induce deletion of CTG.CAG repeats in an orientation-dependent manner in Escherichia coli

The influences of double-strand breaks (DSBs) within a triplet repeat sequence on its genetic instabilities (expansions and deletions) related to hereditary neurological diseases was investigated. Plasmids containing 43 or 70 CTG.CAG repeats or 43 CGG.CCG repeats were linearized in vitro near the center of the repeats and were transformed into parental, RecA-dependent homologous recombination-deficient, or RecBC exonuclease-deficient Escherichia coli. The resulting repair process considerably increased deletion of the repeating sequence compared to the circular DNA controls. Unexpectedly, the orientation of the insert relative to the unidirectional ColE1 origin of replication affected the amount of instability generated during the repair of the DSB. When the CTG strand was the template for lagging-strand synthesis, instability was increased, most markedly in the recA- strain. Results indicated that RecA and/or RecBC might play a role in DSB repair within the triplet repeat. Altering the length, orientation, and sequence composition of the triplet repeat suggested an important role of DNA secondary structures during repair intermediates. Hence, we hypothesize that ColE1 origin-dependent replication was involved during the repair of the DSB. A model is presented to explain the mechanisms of the observed genetic instabilities.     

Nucleotide sequence analysis of cellular flanking regions of intracisternal A-particle gene 81

The cellular nucleotide sequences flanking the mouse intracisternal A-particle gene 81 were determined. The results indicated that they were made of many small oligonucleotide repeats both direct and indirect in orientation. These two different kinds of repeating sequences were often found to be overlap. The overall base composition of this region is relatively A + T rich. The most important feature of the sequences determined was that it consists of several repeated dinucleotide tracts containing a (CA)16 repeating cluster in the 5' end flanking region of one strand and another repeating dinucleotide cluster, (GT)16, in the 3' end flanking region of the same strand of this gene. In addition, the existence of two clusters of 9 continuous 5-bp repeat units, GCTTT, was found in the 3' end flanking region. The possible roles of such repeating sequence were discussed.     

Structure and dynamics in DNA looped domains: CAG triplet repeat sequence dynamics probed by 2-aminopurine fluorescence

The triplet repeat sequence (CAG)n and related triplet repeats are associated with dynamic DNA mutations implicated in a number of debilitating human diseases. To gain insight into the dynamics of the (CAG)n repeat, we have substituted a single 2-aminopurine (2AP) fluorescent base for adenine at select positions within the 18 base looped domain of a (GC)3(CAG)6(GC)3 hairpin oligonucleotide. Using temperature-dependent steady-state fluorescence measurements in combination with time correlated photon counting spectroscopy, we show the conformation and dynamics of the C2APG domains to be strongly dependent on the position of the probe in the looped region. In other words, rather than being a uniform, single stranded loop, the (CAG)6 triplet repeat looped domain exhibits order and dynamics that are position dependent. The 2AP fluorescence dynamics within the C2APG repeat are well described by a 4 component exponential decay model, with lifetimes ranging from 5 ps to 4 ns. Differences in global DNA conformation (duplex, hairpin, single strand), as well as the local position of the probe within the loop of a given hairpin, predominantly are reflected in the relative amplitude rather than the lifetime of the probe. The time dependent 2AP anisotropy in the hairpin (CAG)n loops is sensitive to the position of the fluorescent base, with the fluorescence depolarization of a centrally located 2AP probe within the loop proceeding significantly more slowly than 2AP positioned at the 5'- or 3'-end of the repeat sequence near the loop-stem junction. These results are consistent with segmental motions of the CAG repeat, while also suggesting that the 2AP probe is significantly stacked, possibly even hydrogen bonded, within the partially structured CAG looped domain. Our results characterize the position-dependent and conformation-dependent dynamics and order within (CAG)n triplet repeat DNAs, properties of relevance to the biological mechanisms by which such domains can lead to disease states.     

A most primitive primodial gene as a building block of the genes for the adenylate kinase, F1-ATPase subunits (epsilon and gamma), aminoacyl-tRNA synthetase, and core enzyme subunits

Internal homology units of F1-ATPase epsilon and gamma subunits were searched by computer-aided methods. The epsilon in E. coli (EC) and maize chloroplast (Ch1) was found to consist of three homologous domains, named domains I, II and III (amino acids 1-47, 48-95 and 96-139 for EC). The gamma in E. coli was demonstrated to have at least six homologous domains, tentatively named here domains I-III and V-VII (I = aa 1-23, II = 26-69, III = 71-112, V = 150-192, VI = 196-242, VII = 285-329), with leaving a region IV (113-149) unclassified. Adenylate kinases (AK's) in pig and E. coli were found to have three internal homology units, named I, I' and II (I = aa 1-47, I' = 48-79, II = 80-124 for pig). Statistical evaluations and dot matrix analyses at both base and amino acid sequence levels have confirmed that all of these repeating units, being about 46 amino acids long, are homologous with one another. Of these, epsilon III, II, gamma VII and AK II domains were most conservative and some of them showed homology to core enzyme alpha and an internal repeating unit of tryptophanyl-tRNA synthetase (Trp-RS). Thus these homology unit-encoding gene segments must be relics of a primodial gene.     

The functional origin of bacteriophage f1 DNA replication. Its signals and domains

The origin of DNA replication of bacteriophage f1 functions as a signal, not only for initiation of viral strand synthesis, but also for its termination. Viral (plus) strand synthesis initiates and terminates at a specific site (plus origin) that is recognized and nicked by the viral gene II protein. Mutational analysis of the 5' side (upstream) of the origin of plus strand replication of phage f1 led us to postulate the existence of a set of overlapping functional domains. These included ones for strand nicking, and initiation and termination of DNA synthesis. Mutational analysis of the 3' side (downstream) of the origin has verified the existence of these domains and determined their extent. The results indicate that the f1 "functional origin" can be divided into two domains: (1) a "core region", about 40 nucleotides long, that is absolutely required for plus strand synthesis and contains three distinct but partially overlapping signals, (a) the gene II protein recognition sequence, which is necessary both for plus strand initiation and termination, (b) the termination signal, which extends for eight more nucleotides on the 5' side of the gene II protein recognition sequence, (c) the initiation signal that extends for about ten more nucleotides on the 3' side of the gene II protein recognition sequence; (2) a "secondary region", 100 nucleotides long, required exclusively for plus strand initiation. Disruption of the secondary region does not completely abolish the functionality of the f1 origin but does drastically reduce it (1% residual biological activity). We discuss a possible explanation of the fact that this region can be interrupted (e.g. f1, M13 cloning vectors) by large insertions of foreign DNA without significantly affecting replication.     

Action of S1 nuclease on nicked circular simian virus 40 DNA.

SV40 DNA form II (FO II) containing on average more than one single strand nick per molecule was treated with S₁ nuclease. Linear duplex molecules of unit length (FO III) were generated at enzyme concentrations sufficient to achieve 95% hydrolysis of at least 100 times the amount of single-stranded DNA. Therefore, S₁ nuclease introduces under the described conditions only one double strand break per molecule despite the presence of several single strand nicks.     

RNA-primed initiation sites of DNA replication in the origin region of bacteriophage lambda genome.

Using DNA molecules synthesized in the early stage of lambda phage infection, deoxynucleotides at the transition sites from primer RNA to DNA synthesis have been mapped in the 1.5 kbase area of the lambda phage genome containing the genetically defined replication origin (ori lambda). Sites in the 1-strand (the polarity of the 1-strand is 5' to 3' from the left to the right direction of the lambda phage genetic map) were distributed both inside and outside of the ori lambda, whereas the sites in the r-strand (the strand in the opposite polarity) were mainly distributed more than three hundred nucleotides apart from the ori lambda to the right. A CPuPu sequence was found at -12 to -10 region of transition sites of the r- and the 1-strands in the frequency of 80% and 70%, respectively, and over 60% of the CPuPu sequences were CAG. Properties of the transition sites are discussed in relation to the primer synthesis.