Analysis of the mechanism of DNA recombination using tangles

1995 ◽  
Vol 28 (3) ◽  
pp. 253-313 ◽  
Author(s):  
De Witt Sumners ◽  
Claus Ernst ◽  
Sylvia J. Spengler ◽  
Nicholas R. Cozzarelli
Keyword(s):  
Dna Recombination ◽  
Dna Supercoiling ◽  
Topological Properties ◽  
Circular Dna ◽  
Linear Dna ◽  
Naturally Occurring

The DNA of all organisms has a complex and essential topology. The three topological properties of naturally occurring DNA are supercoiling, catenation, and knotting. Although these properties are denned rigorously only for closed circular DNA, even linear DNA in vivo can have topological properties because it is divided into topologically separate subdomains (Drlica 1987; Roberge & Gasser, 1992). The essentiality of topological properties is demonstrated by the lethal consequence of interfering with topoisomerases, the enzymes that regulate the level of DNA supercoiling and that unlink DNA during its replication (reviewed in Wang, 1991; Bjornsti, 1991; Drlica, 1992; Ullsperger et al. 1995).

scholarly journals Mono-homologous linear DNA recombination by the non-homologous end-joining pathway as a novel and simple gene inactivation method: a proof of concept study inDietziasp. DQ12-45-1b

2018 ◽  
Author(s):  
Shelian Lu ◽  
Yong Nie ◽  
Meng Wang ◽  
Hong-Xiu Xu ◽  
Dong-Ling Ma ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Dna Recombination ◽  
Proof Of Concept ◽  
End Joining ◽  
Circular Dna ◽  
Linear Dna ◽  
Genes Encoding ◽  
Non Homologous End Joining ◽  
Nhej Activity

ABSTRACTNon-homologous end-joining (NHEJ) is critical for genome stability because of its roles in double-strand break repair. Ku and ligase D (LigD) are the crucial proteins in this process, and strains expressing Ku and LigD can cyclize linear DNAin vivo.Herein, we established a proof-of-concept mono-homologous linear DNA recombination for gene inactivation or genome editing by which cyclization of linear DNAin vivoby NHEJ could be used to generate non-replicable circular DNA and could allow allelic exchanges between the circular DNA and the chromosome. We achieved this approach inDietziasp. DQ12-45-1b, which expresses Ku and LigD homologs and presents NHEJ activity. By transforming the strain with a linear DNA mono homolog to the sequence in chromosome, we mutated the genome. This method did not require the screening of suitable plasmids and was easy and time-effective. Bioinformatic analysis showed that more than 20% prokaryotic organisms contain Ku and LigD, suggesting the wide distribution of NHEJ activities. Moreover, theEscherichia colistrain also showed NHEJ activity when the Ku and LigD ofDietziasp. DQ12-45-1b were introduced and expressed in it. Therefore, this method may be a widely applicable genome editing tool for diverse prokaryotic organisms, especially for non-model microorganisms.IMPORTANCEThe non-model gram-positive bacteria lack efficient genetic manipulation systems, but they express genes encoding Ku and LigD. The NHEJ pathway inDietziasp. DQ12-45-1b was evaluated and was used to successfully knockout eleven genes in the genome. Since bioinformatic studies revealed that the putative genes encoding Ku and LigD ubiquitously exist in phylogenetically diverse bacteria and archaea, the mono-homologous linear DNA recombination by the NHEJ pathway could be a potentially applicable genetic manipulation method for diverse non-model prokaryotic organisms.

scholarly journals Single-Homology-Arm Linear DNA Recombination by the Nonhomologous End Joining Pathway as a Novel and Simple Gene Inactivation Method: a Proof-of-Concept Study inDietziasp. Strain DQ12-45-1b

2018 ◽  
Vol 84 (19) ◽  
Author(s):  
Shelian Lu ◽  
Yong Nie ◽  
Meng Wang ◽  
Hong-Xiu Xu ◽  
Dong-Ling Ma ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Dna Recombination ◽  
Nonhomologous End Joining ◽  
Proof Of Concept ◽  
End Joining ◽  
Circular Dna ◽  
Linear Dna ◽  
Genes Encoding ◽  
Nhej Activity

ABSTRACTNonhomologous end joining (NHEJ) is critical for genome stability because of its roles in double-strand break repair. Ku and ligase D (LigD) are the crucial proteins in this process, and strains expressing Ku and LigD can cyclize linear DNAin vivo. Here, we established a proof-of-concept single-homology-arm linear DNA recombination for gene inactivation or genome editing by which cyclization of linear DNAin vivoby NHEJ could be used to generate nonreplicable circular DNA and could allow allelic exchanges between the circular DNA and the chromosome. We achieved this approach inDietziasp. strain DQ12-45-1b, which expresses Ku and LigD homologs and presents NHEJ activity. By transforming the strain with a linear DNA single homolog to the sequence in the chromosome, we mutated the genome. This method did not require the screening of suitable plasmids and was easy and time-effective. Bioinformatic analysis showed that more than 20% of prokaryotic organisms contain Ku and LigD, suggesting the wide distribution of NHEJ activities. Moreover, anEscherichia colistrain also showed NHEJ activity when the Ku and LigD ofDietziasp. DQ12-45-1b were introduced and expressed in it. Therefore, this method may be a widely applicable genome editing tool for diverse prokaryotic organisms, especially for nonmodel microorganisms.IMPORTANCEMany nonmodel Gram-positive bacteria lack efficient genetic manipulation systems, but they express genes encoding Ku and LigD. The NHEJ pathway inDietziasp. DQ12-45-1b was evaluated and was used to successfully knock out 11 genes in the genome. Since bioinformatic studies revealed that the putative genes encoding Ku and LigD ubiquitously exist in phylogenetically diverse bacteria and archaea, the single-homology-arm linear DNA recombination by the NHEJ pathway could be a potentially applicable genetic manipulation method for diverse nonmodel prokaryotic organisms.

scholarly journals Interaction of the Escherichia coli HU Protein with Various Topological Forms of DNA

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1724
Author(s):  
Li Huang ◽  
Zhenfeng Zhang ◽  
Roger McMacken
Keyword(s):  
Dna Supercoiling ◽  
Cellular Level ◽  
Base Pairs ◽  
Supercoiled Dna ◽  
Linear Dna ◽  
Hu Protein ◽  
Binding Preference ◽  
Mass Ratios ◽  
Superhelical Density

E. coli histone-like protein HU has been shown to interact with different topological forms of DNA. Using radiolabeled HU, we examine the effects of DNA supercoiling on HU–DNA interactions. We show that HU binds preferentially to negatively supercoiled DNA and that the affinity of HU for DNA increases with increases in the negative superhelical density of DNA. Binding of HU to DNA is most sensitively influenced by DNA supercoiling within a narrow but physiologically relevant range of superhelicity (σ = −0.06–0). Under stoichiometric binding conditions, the affinity of HU for negatively supercoiled DNA (σ = −0.06) is more than 10 times higher than that for relaxed DNA at physiologically relevant HU/DNA mass ratios (e.g., 1:10). This binding preference, however, becomes negligible at HU/DNA mass ratios higher than 1:2. At saturation, HU binds both negatively supercoiled and relaxed DNA with similar stoichiometries, i.e., 5–6 base pairs per HU dimer. In our chemical crosslinking studies, we demonstrate that HU molecules bound to negatively supercoiled DNA are more readily crosslinked than those bound to linear DNA. At in vivo HU/DNA ratios, HU appears to exist predominantly in a tetrameric form on negatively supercoiled DNA and in a dimeric form on linear DNA. Using a DNA ligase-mediated nick closure assay, we show that approximately 20 HU dimers are required to constrain one negative supercoil on relaxed DNA. Although fewer HU dimers may be needed to constrain one negative supercoil on negatively supercoiled DNA, our results and estimates of the cellular level of HU argue against a major role for HU in constraining supercoils in vivo. We discuss our data within the context of the dynamic distribution of the HU protein in cells, where temporal and local changes of DNA supercoiling are known to take place.

scholarly journals Infection of Ducklings with Virus Particles Containing Linear Double-Stranded Duck Hepatitis B Virus DNA: Illegitimate Replication and Reversion

1998 ◽  
Vol 72 (11) ◽  
pp. 8710-8717 ◽  
Author(s):  
Wengang Yang ◽  
Jesse Summers
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Dna ◽  
Circular Dna ◽  
Virus Particles ◽  
Linear Dna ◽  
Nonhomologous Recombination ◽  
Duck Hepatitis ◽  
B Virus

ABSTRACT Double-stranded linear DNA is synthesized as a minor viral DNA species by all hepadnaviruses. In a previous study (W. Yang and J. Summers, J. Virol. 69:4029–4036, 1995) we showed that virus particles containing linear DNA of the duck hepatitis B virus (DHBV) could initiate an infection of primary duck hepatocytes. In cells infected by linear DNA containing viruses the transcriptional template, covalently closed circular DNA, was formed by circularization of linear DNA by nonhomologous recombination between the two ends. This process was shown to result in viral DNA replication through multiple generations of linear DNA intermediates, a process we called illegitimate replication. In this study we showed that viruses containing linear DHBV DNA produced by engineered insertions in the r sequence, which encodes the 5′ end of the pregenome, could infect hepatocytes in vivo, and these hepatocytes proceeded to carry out illegitimate replication. Nonhomologous recombination quickly produced revertants and partial revertants in which all or part of the insertion was deleted. One such partial revertant that replicated primarily through circular DNA intermediates, but which synthesized elevated levels of linear DNA, could be sustained for several days as the predominant genotype in vivo, but this mutant was eventually displaced by variants showing full reversion to legitimate replication and that synthesized normal low levels of linear DNA. Full revertants did not necessarily contain the wild-type r sequence. The results suggest that the linear DNA produced during DHBV infection initiates cycles of illegitimate replication by generating mutants with altered r sequences. Some r sequence mutants carry out a mixture of legitimate and illegitimate replication that can contribute to elevated production of linear DNA in individual cells.

scholarly journals DNA thermodynamics shape chromosome organization and topology

2013 ◽  
Vol 41 (2) ◽  
pp. 548-553 ◽  
Author(s):  
Andrew A. Travers ◽  
Georgi Muskhelishvili
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Chromosome Organization ◽  
Topological Properties ◽  
Genetic Organization ◽  
And Topology ◽  
Dna Translocases

How much information is encoded in the DNA sequence of an organism? We argue that the informational, mechanical and topological properties of DNA are interdependent and act together to specify the primary characteristics of genetic organization and chromatin structures. Superhelicity generated in vivo, in part by the action of DNA translocases, can be transmitted to topologically sensitive regions encoded by less stable DNA sequences.

scholarly journals Conifers Phytochemicals: A Valuable Forest with Therapeutic Potential

Molecules ◽  
2021 ◽  
Vol 26 (10) ◽  
pp. 3005
Author(s):  
Kanchan Bhardwaj ◽  
Ana Sanches Silva ◽  
Maria Atanassova ◽  
Rohit Sharma ◽  
Eugenie Nepovimova ◽  
...  
Keyword(s):  
Experimental Data ◽  
Potential Role ◽  
Therapeutic Potential ◽  
Fungal Infections ◽  
Cell Damage ◽  
Cancer Growth ◽  
Naturally Occurring ◽  
Antioxidant Effects

Conifers have long been recognized for their therapeutic potential in different disorders. Alkaloids, terpenes and polyphenols are the most abundant naturally occurring phytochemicals in these plants. Here, we provide an overview of the phytochemistry and related commercial products obtained from conifers. The pharmacological actions of different phytochemicals present in conifers against bacterial and fungal infections, cancer, diabetes and cardiovascular diseases are also reviewed. Data obtained from experimental and clinical studies performed to date clearly underline that such compounds exert promising antioxidant effects, being able to inhibit cell damage, cancer growth, inflammation and the onset of neurodegenerative diseases. Therefore, an attempt has been made with the intent to highlight the importance of conifer-derived extracts for pharmacological purposes, with the support of relevant in vitro and in vivo experimental data. In short, this review comprehends the information published to date related to conifers’ phytochemicals and illustrates their potential role as drugs.

scholarly journals UV-induced Hyperphosphorylation of Replication Protein A Depends on DNA Replication and Expression of ATM Protein

2001 ◽  
Vol 12 (5) ◽  
pp. 1199-1213 ◽  
Author(s):  
Gregory G. Oakley ◽  
Lisa I. Loberg ◽  
Jiaqin Yao ◽  
Mary A. Risinger ◽  
Remy L. Yunker ◽  
...  
Keyword(s):  
Dna Replication ◽  
Uv Radiation ◽  
Excision Repair ◽  
Genetic Disorder ◽  
Protein A ◽  
Dna Recombination ◽  
Delayed Response ◽  
Replication Intermediates

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4–8 h) to UV radiation (10–30 J/m2). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

The tra locus of streptomycete plasmid pIJ101 mediates efficient transfer of a circular but not a linear version of the same replicon

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2723-2733 ◽  
Author(s):  
Jing Wang ◽  
Gregg S. Pettis
Keyword(s):  
Conjugal Transfer ◽  
Circular Dna ◽  
Linear Dna ◽  
Double Stranded Dna ◽  
Donor Cells ◽  
Linear Version ◽  
Necessary And Sufficient ◽  
Unique Mechanism ◽  
Original Configuration ◽  
Efficient Transfer

Conjugal transfer of circular plasmids in Streptomyces involves a unique mechanism employing few plasmid-encoded loci and the transfer of double-stranded DNA by an as yet uncharacterized intercellular route. Efficient transfer of the circular streptomycete plasmid pIJ101 requires only two plasmid loci: the pIJ101 tra gene, and as a cis-acting function known as clt. Here, we compared the ability of the pIJ101 transfer apparatus to promote conjugal transfer of circular versus linear versions of the same replicon. While the pIJ101 tra locus readily transferred the circular form of the replicon, the linear version was transferred orders of magnitude less efficiently and all plasmids isolated from the transconjugants were circular, regardless of their original configuration in the donor. Additionally, relatively rare circularization of linear plasmids was detectable in the donor cells, which is consistent with the notion that this event was a prerequisite for transfer by TraB(pIJ101). Linear versions of this same replicon did transfer efficiently, in that configuration, from strains containing the conjugative linear plasmid SLP2. Our data indicate that functions necessary and sufficient for transfer of circular DNA were insufficient for transfer of a related linear DNA molecule. The results here suggest that the conjugation mechanisms of linear versus circular DNA in Streptomyces spp. are inherently different and/or that efficient transfer of linear DNA requires additional components.

scholarly journals Regulation of rat magnocellular neurosecretory system by D-aspartate: evidence for biological role(s) of a naturally occurring free D-amino acid in mammals

2000 ◽  
Vol 167 (2) ◽  
pp. 247-252 ◽  
Author(s):  
H Wang ◽  
H Wolosker ◽  
J Pevsner ◽  
SH Snyder ◽  
DJ Selkoe
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Amino Acid ◽  
Physiological Function ◽  
Neurosecretory System ◽  
Milk Ejection ◽  
Magnocellular Neurons ◽  
Rat Hypothalamus ◽  
Naturally Occurring

Little evidence is available for the physiological function of D-amino acids in species other than bacteria. Here we demonstrate that naturally occurring freed -aspartate (D-Asp) is present in all magnocellular neurons of rat hypothalamus. The levels of this naturally occurring D-amino acid were elevated during lactation and returned to normal thereafter in the magnocellular neurosecretory system, which produces oxytocin, a hormone responsible for milk ejection during lactation. Intraperitoneal injections of D-Asp reproducibly increased oxytocin gene expression and decreased the concentration of circulating oxytocin in vivo. Similar changes were observed in the vasopressin system. These results provide evidence for the role(s) of naturally occurring free D-Asp in mammalian physiology. The findings argue against the conventional concept that only L-stereoisomers of amino acids are functional in higher species.

scholarly journals In Vivo and In Vitro Protein Ligation by Naturally Occurring and Engineered Split DnaE Inteins

PLoS ONE ◽  
2009 ◽  
Vol 4 (4) ◽  
pp. e5185 ◽  
Author(s):  
A. Sesilja Aranko ◽  
Sara Züger ◽  
Edith Buchinger ◽  
Hideo Iwaï
Keyword(s):  
Protein Ligation ◽  
Naturally Occurring ◽  
Vitro Protein
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