The proximity of mini-Mu termini can affect the products of DNA transposition

1994 ◽  
Vol 40 (7) ◽  
pp. 567-575 ◽  
Author(s):  
Michael S. DuBow ◽  
Manon Lalumière
Keyword(s):  
Dna Sequences ◽  
Lytic Cycle ◽  
Temperate Bacteriophage ◽  
Base Pairs ◽  
Dna Transposition ◽  
Double Stranded Dna ◽  
Mu Transposition ◽  
In Cis ◽  
Orientation Parameters

Temperate bacteriophage Mu's 37 kilobase pair (kb) linear, double-stranded DNA requires DNA sequences at both termini in cis to transpose in a duplicative manner and thus propagate its genome during the lytic cycle. To define any spatial or orientation parameters between these sequences, we constructed "isomer" plasmids pMD186 and pMD861 containing identical Mu termini in close (or inverse) orientation 407 base pairs apart, or separated by 9.3 kb of plasmid pSC 101 DNA, respectively. Both mini-Mu plasmids transposed at equal frequencies onto an F′ episome in vivo, in the presence of an induced helper Mu prophage. However, while the vast majority of pMD861 mini-Mu transposition and maturation events were found to be the result of duplicative transposition (cointegrates), virtually all pMD186 transpositions yielded simple linear insertions of the mini-Mu plasmid without concomitant duplication of the mini-Mu. However, the construction of pMD186 mini-Mu plasmid derivatives, containing increased lengths of DNA sequences cloned between the ends of the Mu genome, yielded increasing frequencies of duplicative transposition products. These results suggest that the distance between the Mu termini can affect the final outcome of the transposition reaction.Key words: DNA transposition, phage Mu, cointegrates.

In vitro excision of adeno-associated virus DNA from recombinant plasmids: isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences

1988 ◽  
Vol 8 (6) ◽  
pp. 2513-2522
Author(s):  
J Gottlieb ◽  
N Muzyczka
Keyword(s):  
Dna Sequences ◽  
Base Pairs ◽  
Adeno Associated Virus ◽  
Recombinant Plasmids ◽  
Culture Cells ◽  
Double Stranded Dna ◽  
Enzyme Fraction ◽  
Nuclear Extracts

When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, we isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G.C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat and in some cases as the result of cloning the AAV genome by G.C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.

scholarly journals In vitro excision of adeno-associated virus DNA from recombinant plasmids: isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences.

1988 ◽  
Vol 8 (6) ◽  
pp. 2513-2522 ◽  
Author(s):  
J Gottlieb ◽  
N Muzyczka
Keyword(s):  
Dna Sequences ◽  
Base Pairs ◽  
Adeno Associated Virus ◽  
Recombinant Plasmids ◽  
Culture Cells ◽  
Double Stranded Dna ◽  
Enzyme Fraction ◽  
Nuclear Extracts

When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, we isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G.C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat and in some cases as the result of cloning the AAV genome by G.C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.

scholarly journals Heterogeneous nuclear ribonucleoprotein D0B is a sequence-specificDNA-binding protein

1999 ◽  
Vol 338 (2) ◽  
pp. 417-425 ◽  
Author(s):  
Mate TOLNAY ◽  
Lyudmila A. VERESHCHAGINA ◽  
George C. TSOKOS
Keyword(s):  
Dna Sequences ◽  
Rna Binding ◽  
Binding Protein ◽  
Double Helix ◽  
Physiological Role ◽  
Single Stranded Dna ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Double Stranded Dna ◽  
Nuclear Ribonucleoprotein

Complement receptor 2 (CR2) is important in the regulation of the B lymphocyte response; the regulation of its expression is therefore of central importance. We recently reported that a 42 kDa heterogeneous nuclear ribonucleoprotein (hnRNP) is involved in the transcriptional regulation of the human CR2 gene [Tolnay, Lambris and Tsokos (1997) J. Immunol. 159, 5492–5501]. We cloned the cDNA encoding this protein and found it to be identical with hnRNP D0B, a sequence-specific RNA-binding protein. By using a set of mutated oligonucleotides, we demonstrated that the recombinant hnRNP D0B displays sequence specificity for double-stranded oligonucleotide defined by the CR2 promoter. We conducted electrophoretic mobility-shift assays to estimate the apparent Kd of hnRNP D0B for the double-stranded DNA motif and found it to be 59 nM. Interestingly, hnRNP D0B displayed affinities of 28 and 18 nM for the sense and anti-sense strands of the CR2 promoter-defined oligonucleotide respectively. The significantly greater binding affinity of hnRNP D0B for single-stranded DNA than for double-stranded DNA suggests that the protein might melt the double helix. The intranuclear concentration of sequence-specific protein was estimated to be 250–400 nM, indicating that the protein binds to the CR2 promoter in vivo. Co-precipitation of a complex formed in vivo between hnRNP D0B and the TATA-binding protein demonstrates that hnRNP D0B interacts with the basal transcription apparatus. Our results suggest a new physiological role for hnRNP D0B that involves binding to double- and single-stranded DNA sequences in a specific manner and functioning as a transcription factor.

scholarly journals Transformation of Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo ligation of transforming DNA to mitochondrial DNA sequences

1993 ◽  
Vol 13 (5) ◽  
pp. 2697-2705
Author(s):  
R H Schiestl ◽  
M Dominska ◽  
T D Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Topoisomerase I ◽  
Target Sequence ◽  
Base Pairing ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Fragment

When the yeast Saccharomyces cerevisiae was transformed with DNA that shares no homology to the genome, three classes of transformants were obtained. In the most common class, the DNA was inserted as the result of a reaction that appears to require base pairing between the target sequence and the terminal few base pairs of the transforming DNA fragment. In the second class, no such homology was detected, and the transforming DNA was integrated next to a CTT or GTT in the target; it is likely that these integration events were mediated by topoisomerase I. The final class involved the in vivo ligation of transforming DNA with nucleus-localized linear fragments of mitochondrial DNA.

scholarly journals Hoogsteen base pairs increase the susceptibility of double-stranded DNA to cytotoxic damage

2020 ◽  
Vol 295 (47) ◽  
pp. 15933-15947
Author(s):  
Yu Xu ◽  
Akanksha Manghrani ◽  
Bei Liu ◽  
Honglue Shi ◽  
Uyen Pham ◽  
...  
Keyword(s):  
Dynamic Equilibrium ◽  
Alkylating Agents ◽  
Dimethyl Sulfate ◽  
Dot Blot ◽  
Base Pairs ◽  
Double Stranded Dna ◽  
Genome Wide ◽  
Damage Susceptibility

As the Watson–Crick faces of nucleobases are protected in dsDNA, it is commonly assumed that deleterious alkylation damage to the Watson–Crick faces of nucleobases predominantly occurs when DNA becomes single-stranded during replication and transcription. However, damage to the Watson–Crick faces of nucleobases has been reported in dsDNA in vitro through mechanisms that are not understood. In addition, the extent of protection from methylation damage conferred by dsDNA relative to ssDNA has not been quantified. Watson–Crick base pairs in dsDNA exist in dynamic equilibrium with Hoogsteen base pairs that expose the Watson–Crick faces of purine nucleobases to solvent. Whether this can influence the damage susceptibility of dsDNA remains unknown. Using dot-blot and primer extension assays, we measured the susceptibility of adenine-N1 to methylation by dimethyl sulfate (DMS) when in an A-T Watson–Crick versus Hoogsteen conformation. Relative to unpaired adenines in a bulge, Watson–Crick A-T base pairs in dsDNA only conferred ∼130-fold protection against adenine-N1 methylation, and this protection was reduced to ∼40-fold for A(syn)-T Hoogsteen base pairs embedded in a DNA-drug complex. Our results indicate that Watson–Crick faces of nucleobases are accessible to alkylating agents in canonical dsDNA and that Hoogsteen base pairs increase this accessibility. Given the higher abundance of dsDNA relative to ssDNA, these results suggest that dsDNA could be a substantial source of cytotoxic damage. The work establishes DMS probing as a method for characterizing A(syn)-T Hoogsteen base pairs in vitro and also lays the foundation for a sequencing approach to map A(syn)-T Hoogsteen and unpaired adenines genome-wide in vivo.

scholarly journals HOMOLOGOUS RECOMBINATION IN ESCHERICHIA COLI: DEPENDENCE ON SUBSTRATE LENGTH AND HOMOLOGY

Genetics ◽  
1986 ◽  
Vol 112 (3) ◽  
pp. 441-457 ◽  
Author(s):  
Ping Shen ◽  
Henry V Huang
Keyword(s):  
Dna Sequences ◽  
Wild Type ◽  
Base Pairs ◽  
E Coli ◽  
In Vivo Recombination ◽  
Recombinant Frequency ◽  
Genetic Length ◽  
Efficient Processing ◽  
Low Levels

ABSTRACT We studied the in vivo recombination between homologous DNA sequences cloned in phage lambda and a pBR322-derived plasmid by assaying for the formation of phage-plasmid cointegrates by a single (or an odd number of) reciprocal exchange. (1) Recombination proceeds by the RecBC pathway in wild-type cells and by low levels of a RecF-dependent pathway in recBC  - cells. The RecE pathway appears not to generate phage-plasmid cointegrates. (2) Recombination is linearly dependent on the length of the homologous sequences. In both RecBC and RecF-dependent pathways there is a minimal length, called the minimal efficient processing segment (MEPS), below which recombination becomes inefficient. The length of MEPS is between 23-27 base pairs (bp) and between 44-90 bp for the RecBC- and RecF-dependent pathways, respectively. A model, based on overlapping MEPS, of the correlation of genetic length with physical length is presented. The bases for the different MEPS length of the two pathways are discussed in relationship to the enzymes specific to each pathway. (3) The RecBC and the RecF-dependent pathways are each very sensitive to substrate homology. In wild-type E. coli, reduction of homology from 100% to 90% decreases recombinant frequency over 40-fold. The homology dependence of the RecBC and RecF-dependent pathways are similar. This suggests that a component common to both, probably recA, is responsible for the recognition of homology.

scholarly journals Coarse-Grained Modelling of DNA Plectoneme Pinning in the Presence of Base-Pair Mismatches

2019 ◽  
Author(s):  
Parth Rakesh Desai ◽  
Sumitabha Brahmachari ◽  
John F. Marko ◽  
Siddhartha Das ◽  
Keir C. Neuman
Keyword(s):  
Salt Concentration ◽  
Mechanical Response ◽  
Dna Supercoiling ◽  
Coarse Grained ◽  
Base Pairs ◽  
Mismatched Dna ◽  
Double Stranded Dna ◽  
Theoretical Predictions ◽  
Statistical Mechanical

ABSTRACTDamaged or mismatched DNA bases result in the formation of physical defects in double-stranded DNA. In vivo, defects in DNA must be rapidly and efficiently repaired to maintain cellular function and integrity. Defects can also alter the mechanical response of DNA to bending and twisting constraints, both of which are important in defining the mechanics of DNA supercoiling. Here, we use coarse-grained molecular dynamics (MD) simulation and supporting statistical-mechanical theory to study the effect of mismatched base pairs on DNA supercoiling. Our simulations show that plectoneme pinning at the mismatch site is deterministic under conditions of relatively high force (> 2 pN) and high salt concentration (> 0.5 M NaCl). Under physiologically relevant conditions of lower force (0.3 pN) and lower salt concentration (0.2 M NaCl), we find that plectoneme pinning becomes probabilistic and the pinning probability increases with the mismatch size. These findings are in line with experimental observations. The simulation framework, validated with experimental results and supported by the theoretical predictions, provides a way to study the effect of defects on DNA supercoiling and the dynamics of supercoiling in molecular detail.

scholarly journals Transformation of Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo ligation of transforming DNA to mitochondrial DNA sequences.

1993 ◽  
Vol 13 (5) ◽  
pp. 2697-2705 ◽  
Author(s):  
R H Schiestl ◽  
M Dominska ◽  
T D Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Topoisomerase I ◽  
Target Sequence ◽  
Base Pairing ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Fragment

When the yeast Saccharomyces cerevisiae was transformed with DNA that shares no homology to the genome, three classes of transformants were obtained. In the most common class, the DNA was inserted as the result of a reaction that appears to require base pairing between the target sequence and the terminal few base pairs of the transforming DNA fragment. In the second class, no such homology was detected, and the transforming DNA was integrated next to a CTT or GTT in the target; it is likely that these integration events were mediated by topoisomerase I. The final class involved the in vivo ligation of transforming DNA with nucleus-localized linear fragments of mitochondrial DNA.

scholarly journals DNA sequences required for specific and efficient initiation of transcription at the polyoma virus early promoter.

1982 ◽  
Vol 2 (7) ◽  
pp. 737-751 ◽  
Author(s):  
P Jat ◽  
U Novak ◽  
A Cowie ◽  
C Tyndall ◽  
R Kamen
Keyword(s):  
Dna Sequences ◽  
In Vitro Transcription ◽  
Polyoma Virus ◽  
Tata Box ◽  
Deletion Mutants ◽  
Base Pairs ◽  
Transcription Analysis ◽  
In Vivo Expression

The 5'-flanking DNA sequences involved in the specific and efficient transcription of the polyoma virus early region have been investigated. Sequence requirements for efficient in vivo expression differed from those in vitro. Deletion of DNA located between 200 and 400 base pairs before the principal cap sites severely inhibited in vivo expression as measured by transformation ability, but did not affect in vitro transcription. Viable deletion mutants which lack the principal cap sites and the "TATA" box were very poor templates for in vitro transcription. Analysis of other deletion mutants in vitro demonstrated that no specific sequences more than 46 base pairs before the cap sites were important. Removal of the TATA box reduced in vitro transcriptional efficiency but did not alter the initiation sites. The synthesis of transcripts with abnormal 5' termini did not occur in vitro until sequence between the TATA box and the normal cap sites was also deleted. We further observed a nonspecific requirement for 90 to 100 base pairs of DNA 5' to the cap site for optimal transcription of DNA fragments in vitro.

DNA sequences required for specific and efficient initiation of transcription at the polyoma virus early promoter

1982 ◽  
Vol 2 (7) ◽  
pp. 737-751
Author(s):  
P Jat ◽  
U Novak ◽  
A Cowie ◽  
C Tyndall ◽  
R Kamen
Keyword(s):  
Dna Sequences ◽  
In Vitro Transcription ◽  
Polyoma Virus ◽  
Tata Box ◽  
Deletion Mutants ◽  
Base Pairs ◽  
Transcription Analysis ◽  
In Vivo Expression

The 5'-flanking DNA sequences involved in the specific and efficient transcription of the polyoma virus early region have been investigated. Sequence requirements for efficient in vivo expression differed from those in vitro. Deletion of DNA located between 200 and 400 base pairs before the principal cap sites severely inhibited in vivo expression as measured by transformation ability, but did not affect in vitro transcription. Viable deletion mutants which lack the principal cap sites and the "TATA" box were very poor templates for in vitro transcription. Analysis of other deletion mutants in vitro demonstrated that no specific sequences more than 46 base pairs before the cap sites were important. Removal of the TATA box reduced in vitro transcriptional efficiency but did not alter the initiation sites. The synthesis of transcripts with abnormal 5' termini did not occur in vitro until sequence between the TATA box and the normal cap sites was also deleted. We further observed a nonspecific requirement for 90 to 100 base pairs of DNA 5' to the cap site for optimal transcription of DNA fragments in vitro.

Sign in / Sign up

Export Citation Format

Share Document