scholarly journals The specificity of topoisomerase-mediated DNA cleavage defines acridine-induced frameshift specificity within a hotspot in bacteriophage T4.

Genetics ◽  
1991 ◽  
Vol 127 (3) ◽  
pp. 453-462 ◽  
Author(s):  
M Masurekar ◽  
K N Kreuzer ◽  
L S Ripley
Keyword(s):  
Dna Sequence ◽  
Dna Cleavage ◽  
Frameshift Mutation ◽  
Bacteriophage T4 ◽  
Precise Location ◽  
Frameshift Mutations ◽  
Phosphodiester Bond ◽  
Frameshift Mutagenesis

Abstract Acridine-induced frameshift mutations in bacteriophage T4 occur at the precise location in the DNA at which acridines stimulate DNA cleavage by the T4-encoded type II topoisomerase in vitro. The mutations are duplications or deletions that begin precisely at the broken phosphodiester bond. In vivo, acridine-induced frameshift mutagenesis is reduced nearly to background levels when the topoisomerase is genetically inactivated. These observations are consistent with a model in which cleaved DNA, induced by the topoisomerase and acridine, serves as the substrate for the production of frameshift mutations at the same site. Our model predicts that the specificity and frequency of cleavage direct the specificity and frequency of mutagenesis. This prediction was tested by examining the influence of DNA sequence changes on topoisomerase-mediated cleavage and on mutagenesis in the T4 rIIB gene. The model successfully predicted the results. When DNA sequence changes altered the position of acridine-induced, topoisomerase-mediated DNA cleavage in vitro, frameshift mutations were found at the new positions. DNA sequence changes that strongly decreased in vitro cleavage also reduced mutagenesis at that site. These results demonstrate that acridine-induced frameshift mutation specificity is directed by the characteristics of the acridine-topoisomerase reaction and do not suggest that slipped pairing in repeated sequences plays a major role in acridine-induced frameshifts in bacteriophage T4.

scholarly journals Identification of a Mutant DNA Polymerase δ in Saccharomyces cerevisiae With an Antimutator Phenotype for Frameshift Mutations

Genetics ◽  
2001 ◽  
Vol 158 (1) ◽  
pp. 177-186 ◽  
Author(s):  
Michalis I Hadjimarcou ◽  
Robert J Kokoska ◽  
Thomas D Petes ◽  
Linda J Reha-Krantz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Bacteriophage T4 ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Base Substitution ◽  
Msh2 Gene ◽  
Frameshift Mutations ◽  
Frameshift Mutagenesis

Abstract We propose that a β-turn-β structure, which plays a critical role in exonucleolytic proofreading in the bacteriophage T4 DNA polymerase, is also present in the Saccharomyces cerevisiae DNA pol δ. Site-directed mutagenesis was used to test this proposal by introducing a mutation into the yeast POL3 gene in the region that encodes the putative β-turn-β structure. The mutant DNA pol δ has a serine substitution in place of glycine at position 447. DNA replication fidelity of the G447S-DNA pol δ was determined in vivo by using reversion and forward assays. An antimutator phenotype for frameshift mutations in short homopolymeric tracts was observed for the G447S-DNA pol δ in the absence of postreplication mismatch repair, which was produced by inactivation of the MSH2 gene. Because the G447S substitution reduced frameshift but not base substitution mutagenesis, some aspect of DNA polymerase proofreading appears to contribute to production of frameshifts. Possible roles of DNA polymerase proofreading in frameshift mutagenesis are discussed.

Targeted chemical nucleases have a wide range of untapped applications in biological fields, including gene therapy

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Metal Complex ◽  
Dna Cleavage ◽  
Therapeutic Potential ◽  
Chemical Reactivity ◽  
Major Groove ◽  
Sequence Recognition ◽  
Wide Range ◽  
Chemical Nucleases

A feasible alternative to state-of-the-art enzymatic nucleases was created by regulating the cleavage activity of metal complexes using (covalent or non-covalent) homing agents. Targeted AMNs, unlike enzymatic nucleases, break DNA by an oxidative mechanism and can therefore permanently knock off genes. Compared to larger enzymatic nucleases, the modest size of the metal complex may aid cellular transfection. Furthermore, the painstaking construction of the sequence-specific probe permits a metal complex to be directed to dsDNA's minor or major groove. To direct the chemical reactivity of several small-molecule compounds to dsDNA's minor groove, covalently bonded polyamide samples were used. PNA and DNA were also used to construct antisense and antigen hybrids, with Watson–Crick or Hoogsteen base pairing with major groove nucleobases giving sequence recognition. Click chemistry created chimeric AMN-TFOs with desirable focused effects and negligible off-target cleavage. Clip-Phen-modified TFOs, 230 polypyridyl-modified TFOs, 232 and intercalating phenanthrene-modified TFOs are three contemporary instances of copper AMN–TFOs. All three systems have distinct advantages in maintaining the desired 2:1 phenthroline/copper ratio for DNA cleavage (clip-Phen TFOs), caging the copper center and facilitating efficient ROS-mediated strand scission (polypyridyl-modified TFO) and improving triplex stability (polypyridyl-modified TFO) (phenanthrene-TFOs). Cerium (IV)/EDTA complexes, recently shown to bind and hydrolytically cleave ssDNA/dsDNA junctions and used in conjunction with PNA to successfully introduce genome changes in vitro and in vivo, are another important class of targeted chemical nucleases. The chemical reactivity and wide flexibility of metal complex design, combined with their coupling to sequence specific samples for directed applications, show that these compounds have a wide range of untapped applications in biological fields such as chemotherapy, protein engineering, DNA footprinting, and gene editing. Parallel advancements in cell and tissue targeting will be essential to maximise their therapeutic potential, either by using specific ligands or creating new targeting modalities.

scholarly journals Mutational analysis of the mRNA operator for T4 DNA polymerase.

Genetics ◽  
1991 ◽  
Vol 128 (2) ◽  
pp. 203-213 ◽  
Author(s):  
M D Andrake ◽  
J D Karam
Keyword(s):  
Dna Polymerase ◽  
Mutational Analysis ◽  
Bacteriophage T4 ◽  
Spatial Arrangement ◽  
Loop Sequence ◽  
Shine Dalgarno Sequence ◽  
In Vitro Effects ◽  
Rna Hairpin

Abstract Biosynthesis of bacteriophage T4 DNA polymerase is autogenously regulated at the translational level. The enzyme, product of gene 43, represses its own translation by binding to its mRNA 5' to the initiator AUG at a 36-40 nucleotide segment that includes the Shine-Dalgarno sequence and a putative RNA hairpin structure consisting of a 5-base-pair stem and an 8-base loop. We constructed mutations that either disrupted the stem or altered specific loop residues of the hairpin and found that many of these mutations, including single-base changes in the loop sequence, diminished binding of purified T4 DNA polymerase to its RNA in vitro (as measured by a gel retardation assay) and derepressed synthesis of the enzyme in vivo (as measured in T4 infections and by recombinant-plasmid-mediated expression). In vitro effects, however, were not always congruent with in vivo effects. For example, stem pairing with a sequence other than wild-type resulted in normal protein binding in vitro but derepression of protein synthesis in vivo. Similarly, a C----A change in the loop had a small effect in vitro and a strong effect in vivo. In contrast, an A----U change near the base of the hairpin that was predicted to increase the length of the base-paired stem had small effects both in vitro and in vivo. The results suggest that interaction of T4 DNA polymerase with its structured RNA operator depends on the spatial arrangement of specific nucleotide residues and is subject to modulation in vivo.

scholarly journals CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting

Gene Therapy ◽  
2015 ◽  
Vol 23 (1) ◽  
pp. 108-112 ◽  
Author(s):  
D G Courtney ◽  
J E Moore ◽  
S D Atkinson ◽  
E Maurizi ◽  
E H A Allen ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Specific Targeting

Simian virus 40 major late promoter: an upstream DNA sequence required for efficient in vitro transcription

1984 ◽  
Vol 4 (1) ◽  
pp. 133-141
Author(s):  
J Brady ◽  
M Radonovich ◽  
M Thoren ◽  
G Das ◽  
N P Salzman
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Simian Virus 40 ◽  
Promoter Sequence ◽  
In Vitro Transcription ◽  
Simian Virus ◽  
Upstream Promoter ◽  
Upstream Promoter Sequence

We have previously identified an 11-base DNA sequence, 5'-G-G-T-A-C-C-T-A-A-C-C-3' (simian virus 40 [SV40] map position 294 to 304), which is important in the control of SV40 late RNA expression in vitro and in vivo (Brady et al., Cell 31:625-633, 1982). We report here the identification of another domain of the SV40 late promoter. A series of mutants with deletions extending from SV40 map position 0 to 300 was prepared by nuclease BAL 31 treatment. The cloned templates were then analyzed for efficiency and accuracy of late SV40 RNA expression in the Manley in vitro transcription system. Our studies showed that, in addition to the promoter domain near map position 300, there are essential DNA sequences between nucleotide positions 74 and 95 that are required for efficient expression of late SV40 RNA. Included in this SV40 DNA sequence were two of the six GGGCGG SV40 repeat sequences and an 11-nucleotide segment which showed strong homology with the upstream sequences required for the efficient in vitro and in vivo expression of the histone H2A gene. This upstream promoter sequence supported transcription with the same efficiency even when it was moved 72 nucleotides closer to the major late cap site. In vitro promoter competition analysis demonstrated that the upstream promoter sequence, independent of the 294 to 304 promoter element, is capable of binding polymerase-transcription factors required for SV40 late gene transcription. Finally, we show that DNA sequences which control the specificity of RNA initiation at nucleotide 325 lie downstream of map position 294.

A MAJOR ROLE FOR BACTERIOPHAGE T4 DNA POLYMERASE IN FRAMESHIFT MUTAGENESIS

Genetics ◽  
1983 ◽  
Vol 103 (3) ◽  
pp. 353-366
Author(s):  
Lynn S Ripley ◽  
Nadja B Shoemaker
Keyword(s):  
Dna Polymerase ◽  
Dna Sequences ◽  
Frameshift Mutation ◽  
Bacteriophage T4 ◽  
Base Substitution ◽  
Temperature Sensitive ◽  
Polymerase Gene ◽  
Frameshift Mutagenesis ◽  
Substitution Mutations ◽  
Polymerase Mutants

ABSTRACT T4 DNA polymerase strongly influences the frequency and specificity of frameshift mutagenesis. Fifteen of 19 temperature-sensitive alleles of the DNA polymerase gene substantially influenced the reversion frequencies of frameshift mutations measured in the T4 rII genes. Most polymerase mutants increased frameshift frequencies, but a few alleles (previously noted as antimutators for base substitution mutations) decreased the frequencies of certain frameshifts while increasing the frequencies of others. The various patterns of enhanced or decreased frameshift mutation frequencies suggest that T4 DNA polymerase is likely to play a variety of roles in the metabolic events leading to frameshift mutation. A detailed genetic study of the specificity of the mutator properties of three DNA polymerase alleles (tsL56, tsL98 and tsL88) demonstrated that each produces a distinctive frameshift spectrum. Differences in frameshift frequencies at similar DNA sequences within the rII genes, the influence of mutant polymerase alleles on these frequencies, and the presence or absence of the dinucleotide sequence associated with initiation of Okazaki pieces at the frameshift site has led us to suggest that the discontinuities associated with discontinuous DNA replication may contribute to spontaneous frameshift mutation frequencies in T4.

Transcription of the bacteriophage T4 template. Detailed comparison of in vitro and in vivo transcripts

Biochemistry ◽  
1970 ◽  
Vol 9 (6) ◽  
pp. 1300-1309 ◽  
Author(s):  
Edward N. Brody ◽  
E. Peter Geiduschek
Keyword(s):  
Bacteriophage T4 ◽  
Detailed Comparison

scholarly journals An Antitumor Drug-Induced Topoisomerase Cleavage Complex Blocks a Bacteriophage T4 Replication Fork In Vivo

2000 ◽  
Vol 20 (2) ◽  
pp. 594-603 ◽  
Author(s):  
George Hong ◽  
Kenneth N. Kreuzer
Keyword(s):  
Dna Cleavage ◽  
Cleavage Site ◽  
Replication Fork ◽  
Bacteriophage T4 ◽  
Antitumor Drug ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Drug Induced ◽  
Cleavage Complex

ABSTRACT Many antitumor and antibacterial drugs inhibit DNA topoisomerases by trapping covalent enzyme-DNA cleavage complexes. Formation of cleavage complexes is important for cytotoxicity, but evidence suggests that cleavage complexes themselves are not sufficient to cause cell death. Rather, active cellular processes such as transcription and/or replication are probably necessary to transform cleavage complexes into cytotoxic lesions. Using defined plasmid substrates and two-dimensional agarose gel analysis, we examined the collision of an active replication fork with an antitumor drug-trapped cleavage complex. Discrete DNA molecules accumulated on the simple Y arc, with branch points very close to the topoisomerase cleavage site. Accumulation of the Y-form DNA required the presence of a topoisomerase cleavage site, the antitumor drug, the type II topoisomerase, and a T4 replication origin on the plasmid. Furthermore, all three arms of the Y-form DNA were replicated, arguing strongly that these are trapped replication intermediates. The Y-form DNA appeared even in the absence of two important phage recombination proteins, implying that Y-form DNA is the result of replication rather than recombination. This is the first direct evidence that a drug-induced topoisomerase cleavage complex blocks the replication fork in vivo. Surprisingly, these blocked replication forks do not contain DNA breaks at the topoisomerase cleavage site, implying that the replication complex was inactivated (at least temporarily) and that topoisomerase resealed the drug-induced DNA breaks. The replication fork may behave similarly at other types of DNA lesions, and thus cleavage complexes could represent a useful (site-specific) model for chemical- and radiation-induced DNA damage.

scholarly journals c-Jun/c-Fos heterodimers regulate cellular genes via a newly identified class of methylated DNA sequence motifs

2013 ◽  
Vol 42 (5) ◽  
pp. 3059-3072 ◽  
Author(s):  
Montse Gustems ◽  
Anne Woellmer ◽  
Ulrich Rothbauer ◽  
Sebastian H. Eck ◽  
Thomas Wieland ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Sequence ◽  
Epigenetic Silencing ◽  
Activator Protein 1 ◽  
Sequence Motifs ◽  
Promoter Regions ◽  
Barr Virus ◽  
Dna Sequence Motifs

Abstract CpG methylation in mammalian DNA is known to interfere with gene expression by inhibiting the binding of transactivators to their cognate sequence motifs or recruiting proteins involved in gene repression. An Epstein–Barr virus-encoded transcription factor, Zta, was the first example of a sequence-specific transcription factor that preferentially recognizes and selectively binds DNA sequence motifs with methylated CpG residues, reverses epigenetic silencing and activates gene transcription. The DNA binding domain of Zta is homologous to c-Fos, a member of the cellular AP-1 (activator protein 1) transcription factor family, which regulates cell proliferation and survival, apoptosis, transformation and oncogenesis. We have identified a novel AP-1 binding site termed meAP-1, which contains a CpG dinucleotide. If methylated, meAP-1 sites are preferentially bound by the AP-1 heterodimer c-Jun/c-Fos in vitro and in cellular chromatin in vivo. In activated human primary B cells, c-Jun/c-Fos locates to these methylated elements in promoter regions of transcriptionally activated genes. Reminiscent of the viral Zta protein, c-Jun/c-Fos is the first identified cellular member of the AP-1 family of transactivators that can induce expression of genes with methylated, hence repressed promoters, reversing epigenetic silencing.

Sign in / Sign up

Export Citation Format

Share Document