Left-Handed DNA in Vivo

Jaworski, Adam; Hsieh, Wang-Ting; Blaho, John A.; Larson, Jacquelynn E.; Wells, Robert D.

doi:10.1126/science.3313728

Cited by 213 publications

(118 citation statements)

References 36 publications

Supporting

Mentioning

115

Contrasting

Order By: Relevance

“…Thus, the non-identical sequences of these two vectors enabled our focus on the potential effects of cloned tracts of different TRS on recombination. Prior investigations (22) revealed the stable cotransformation of derivatives of these two plasmids.…”

Section: Resultsmentioning

confidence: 99%

Genetic Instabilities in (CTG·CAG) Repeats Occur by Recombination

Jakupciak¹,

Wells

1999

Journal of Biological Chemistry

Self Cite

117

View full text Add to dashboard Cite

The expansion of triplet repeat sequences (TRS) associated with hereditary neurological diseases is believed from prior studies to be due to DNA replication. This report demonstrates that the expansion of (CTG⅐CAG) n in vivo also occurs by homologous recombination as shown by biochemical and genetic studies. A two-plasmid recombination system was established in Escherichia coli with derivatives of pUC19 (harboring the ampicillin resistance gene) and pACYC184 (harboring the tetracycline resistance gene). The derivatives contained various triplet repeat inserts ((CTG⅐CAG), (CGG⅐CCG), (GAA⅐TTC), (GTC⅐GAC), and (GTG⅐CAC)) of different lengths, orientations, and extents of interruptions and a control non-repetitive sequence. The availability of the two drug resistance genes and of several unique restriction sites on the plasmids enabled rigorous genetic and biochemical analyses. The requirements for recombination at the TRS include repeat lengths >30, the presence of CTG⅐CAG on both plasmids, and recA and recBC. Sequence analyses on a number of DNA products isolated from individual colonies directly demonstrated the crossing-over and expansion of the homologous CTG⅐CAG regions. Furthermore, inversion products of the type [(CTG) 13 (CAG) 67 ]⅐[(CTG) 67 (CAG) 13 ] were isolated as the apparent result of "illegitimate" recombination events on intrahelical pseudoknots. This work establishes the relationships between CTG⅐CAG sequences, multiple fold expansions, genetic recombination, formation of new recombinant DNA products, and the presence of both drug resistance genes. Thus, if these reactions occur in humans, unequal crossing-over or gene conversion may also contribute to the expansions responsible for anticipation associated with several hereditary neurological syndromes.Genetic instabilities (expansions and deletions) of triplet repeat sequences (TRS) 1 ((CTG⅐CAG), (CGG⅐CCG), or (GAA⅐TTC)) are a hallmark of certain hereditary neurological diseases (1, 2). Numerous workers in human genetics have proposed DNA replication, gene conversion, recombination, and related processes as the mechanism(s) responsible for these alterations in repeat sequence lengths. Subsequent in vivo studies in genetically tractable systems (1, 3) and in vitro investigations (4) have demonstrated expansions and deletions during DNA replication, probably by slipped strand misalignment due to preferential hairpin formation of TRS. Similar molecular studies in vivo on gene conversion and recombination are lacking.Several human genetic studies on patient materials reported haplotype analyses, especially related to myotonic dystrophy (DM) and the fragile X syndrome, which implicated gene conversion and/or unequal crossing-over (types of recombination) to genetic instabilities. In the first report, Korneluk and coworkers (5, 6) proposed unequal crossing-over (5) and gene conversion (6) as the mechanisms responsible for the expansions and deletions observed in the (CTG⅐CAG) mutation during DM transmission. This conclusion was derived from haplotype a...

show abstract

Section: Resultsmentioning

confidence: 99%

Genetic Instabilities in (CTG·CAG) Repeats Occur by Recombination

Jakupciak¹,

Wells

1999

Journal of Biological Chemistry

Self Cite

117

View full text Add to dashboard Cite

show abstract

“…The pACYC184 plasmid contains the chloramphenicol resistance gene and the p15A origin of replication (40). pACYC184 can co-exist in E. coli with pBR322 and can be replicated in the absence of the protein (27,28,41).…”

Section: Expermental Proceduresmentioning

confidence: 99%

Long CTG·CAG Repeats from Myotonic Dystrophy Are Preferred Sites for Intermolecular Recombination

Pluciennik

Iyer

Напиерала

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Homologous recombination was shown to enable the expansion of CTG⅐CAG repeat sequences. Other prior investigations revealed the involvement of replication and DNA repair in these genetic instabilities. Here we used a genetic assay to measure the frequency of homologous intermolecular recombination between two CTG⅐CAG tracts. When compared with non-repeating sequences of similar lengths, long (CTG⅐CAG) n repeats apparently recombine with an ϳ60-fold higher frequency. Sequence polymorphisms that interrupt the homogeneity of the CTG⅐CAG repeat tracts reduce the apparent recombination frequency as compared with the pure uninterrupted repeats. The orientation of the repeats relative to the origin of replication strongly influenced the apparent frequency of recombination. This suggests the involvement of DNA replication in the recombination process of triplet repeats. We propose that DNA polymerases stall within the CTG⅐CAG repeat tracts causing nicks or double-strand breaks that stimulate homologous recombination. The recombination process is RecA-dependent.Micro-and minisatellite instability has been associated with human genetic diseases (1-4). Approximately 14 hereditary neurological diseases are caused by the genetic instabilities of triplet repeat sequences (TRS) 1 in or near relevant genes (reviewed in Ref. 1). Long tracts of repeating CTG⅐CAG sequences are responsible for myotonic dystrophy and several other diseases. Normal individuals have 5-37 repeats in the myotonic dystrophy protein kinase gene, whereas the mutations (expansions) can be in the range of 50 -3000 repeats. Thus, an explosive allele length change during intergenerational transmission can occur in this autosomal dominant disease, thus causing an increase in the severity of the disease and a decrease of the age at onset (anticipation).The mechanisms of genetic instabilities have been widely investigated in the past 6 years (1). DNA replication (5-8) and repair including methyl-directed mismatch repair (9 -11), nucleotide excision repair (12), DNA polymerase III exonucleolytic proofreading (13), and double-strand break repair (14) have been implicated.Repetitive sequences promote homologous recombination in prokaryotic as well as eukaryotic systems, presumably by virtue of forming unusual DNA secondary structures (15)(16)(17)(18)(19)(20)(21)(22). In fact, homologous recombination has been implicated in the instability of repetitive sequences (23-26). Similar findings have also been made with CTG⅐CAG repeats using a two-plasmid system in Escherichia coli (27,28). Multiple fold expansions, deletions, and the exchange of point mutations between tracts were found in this system; these events were dependent on the presence of TRS tracts on both plasmids, CTG⅐CAG repeat lengths longer than 30, and a functional recA gene.Previously (29), it was proposed that CTG⅐CAG tracts might function as recombination hot spots in the bovine genome. More recently, Young et al. (30) surveyed the genome of Saccharomyces cerevisiae and suggested that these repeats cou...

show abstract

“…Left-handed DNA (Z-DNA) is a structural alternative to right-handed DNA (B-DNA) that has been well characterized in vitro (14,17,21). Recently, it was demonstrated that Z-DNA can exist in living Escherichia coli cells and that a given sequence can coexist in the Z form and the B form in the same cell (7,8,15,22,23). The in vivo B-Z equilibrium is influenced by active biological processes (like transcription, replication, supercoil and toroid formations, and DNAprotein interactions).…”

mentioning

confidence: 99%

“…The first assay was based on the observation that a recognition sequence (GAATTC) is not methylated by the corresponding bacterial methyltransferase (M EcoRI) when this site is near or inside a left-handed helix, whereas other EcoRI sites in the same plasmid molecule were completely methylated (8,23). The presence of a Z structure around the unmethylated sites was confirmed by a variety of chemical, enzymatic, and topological approaches (14,16,17,21,24 On this basis, the formation of Z-DNA inside growing bacterial cells could be analyzed by a linking-number assay.…”

mentioning

confidence: 99%

Cytosine methylation enhances Z-DNA formation in vivo

1990

View full text Add to dashboard Cite

The influence of cytosine methylation on the supercoil-stabilized B-Z equilibrium in Escherichia coli was analyzed by two independent assays. Both the M EcoRI inhibition assay and the linking-number assay have been used previously to establish that dC-dG segments of sufficient lengths can exist as left-handed helices in vivo. A series of dC-dG plasmid inserts with Z-form potential, ranging in length from 14 to 74 base pairs, was investigated. Complete methylation of cytosine at all HhaI sites, including the inserts, was obtained by coexpression of the HhaI methyltransferase (M -HhaI) in cells also carrying a dC-dG-containing plasmid. Both assays showed that for all lengths of dC-dG inserts, the relative amounts of B and Z helices were shifted to more Z-DNA in the presence of M -HhaI than in the absence of M HhaI. These results indicate that cytosine methylation enhances the formation of Z-DNA helices at the superhelix density present in E. coli. The B-Z equilibrium, in combination with site-specific base methylation, may constitute a concerted mechanism for the modulation of DNA topology and DNA-protein interactions.DNA structural microheterogeneity is believed to play a fundamental role in genetic regulatory mechanisms (16,20). Left-handed DNA (Z-DNA) is a structural alternative to right-handed DNA (B-DNA) that has been well characterized in vitro (14,17,21). Recently, it was demonstrated that Z-DNA can exist in living Escherichia coli cells and that a given sequence can coexist in the Z form and the B form in the same cell (7,8,15,22,23). The in vivo B-Z equilibrium is influenced by active biological processes (like transcription, replication, supercoil and toroid formations, and DNAprotein interactions).There is evidence that cytosine methylation may have an important signaling function in eucaryotic gene expression (4, 11). The hypomethylation of gene regulatory regions is often connected to an expressed state of those genes, whereas silent genes are generally overmethylated. In vitro, cytosine methylation also facilitates the B-to-Z transition in synthetic linear DNA polymers (1, 2, 5) and in certain sequences in supercoiled recombinant plasmids (10,25).In previous studies, we employed two independent assays for structural analyses under in vivo conditions. The first assay was based on the observation that a recognition sequence (GAATTC) is not methylated by the corresponding bacterial methyltransferase (M EcoRI) when this site is near or inside a left-handed helix, whereas other EcoRI sites in the same plasmid molecule were completely methylated (8,23). The presence of a Z structure around the unmethylated sites was confirmed by a variety of chemical, enzymatic, and topological approaches (14,16,17,21,24 On this basis, the formation of Z-DNA inside growing bacterial cells could be analyzed by a linking-number assay. The B-to-Z transition inside E. coli cells results in the relaxation of a number of supercoils, in proportion to the length of the Z-region involved. This process causes DNA gyrase to decrease...

show abstract

Left-Handed DNA in Vivo

Cited by 213 publications

References 36 publications

Genetic Instabilities in (CTG·CAG) Repeats Occur by Recombination

Genetic Instabilities in (CTG·CAG) Repeats Occur by Recombination

Long CTG·CAG Repeats from Myotonic Dystrophy Are Preferred Sites for Intermolecular Recombination

Cytosine methylation enhances Z-DNA formation in vivo

Contact Info

Product

Resources

About