scholarly journals A DNA-binding protein from Ustilago maydis prefers duplex DNA without chain interruptions.

1983 ◽  
Vol 3 (4) ◽  
pp. 605-612 ◽  
Author(s):  
J R Rusche ◽  
W K Holloman
Keyword(s):  
Drosophila Melanogaster ◽  
Satellite Dna ◽  
Binding Assay ◽  
Ustilago Maydis ◽  
Dna Binding Protein ◽  
Double Strand Breaks ◽  
Duplex Dna ◽  
Plasmid Pbr322 ◽  
Strand Breaks ◽  
Dna Strand

Using a nitrocellulose filter binding assay, we have partially purified a protein from mitotic cells of Ustilago maydis that binds preferentially to covalently closed circular duplex DNA. DNA containing single- or double-strand breaks is bound poorly by the protein. Once formed, the DNA-protein complex is stable, resisting dissociation in high salt. However, when a DNA strand is broken, the complex appears to dissociate. The protein binds equally well to form I DNA of phi X174 or the plasmid pBR322, but has a higher affinity for a hybrid plasmid containing a cloned region of Drosophila melanogaster satellite DNA.

A DNA-binding protein from Ustilago maydis prefers duplex DNA without chain interruptions

1983 ◽  
Vol 3 (4) ◽  
pp. 605-612
Author(s):  
J R Rusche ◽  
W K Holloman
Keyword(s):  
Drosophila Melanogaster ◽  
Satellite Dna ◽  
Binding Assay ◽  
Ustilago Maydis ◽  
Dna Binding Protein ◽  
Double Strand Breaks ◽  
Duplex Dna ◽  
Plasmid Pbr322 ◽  
Strand Breaks ◽  
Dna Strand

Using a nitrocellulose filter binding assay, we have partially purified a protein from mitotic cells of Ustilago maydis that binds preferentially to covalently closed circular duplex DNA. DNA containing single- or double-strand breaks is bound poorly by the protein. Once formed, the DNA-protein complex is stable, resisting dissociation in high salt. However, when a DNA strand is broken, the complex appears to dissociate. The protein binds equally well to form I DNA of phi X174 or the plasmid pBR322, but has a higher affinity for a hybrid plasmid containing a cloned region of Drosophila melanogaster satellite DNA.

scholarly journals Fragmentation of Plasmid DNA Produced by Gamma Radiation: A Theoretical Approach

2012 ◽  
Vol 2012 ◽  
pp. 1-6
Author(s):  
R. A. S. Silva ◽  
J. D. T. Arruda-Neto ◽  
L. Nieto
Keyword(s):  
Plasmid Dna ◽  
Fragment Size ◽  
Power Laws ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Starting Point ◽  
Dna Strands ◽  
High Let ◽  
Fragment Distribution ◽  
Dna Strand

Breaks in DNA, resulting in fragmented parts, can be produced by ionizing radiation which, in turn, is the starting point in the search for novel physical aspects of DNA strands. Double-strand breaks in particular cause disruption of the DNA strand, splitting it into several fragments. In order to study effects produced by radiation in plasmid DNA, a new simple mechanical model for this molecule is proposed. In this model, a Morse-like potential and a high-LET component are used to describe the DNA-radiation interaction. Two power laws, used to fit results of the model, suggest that, firstly, distribution of fragment size is nonextensive and, secondly, that a transition phase is present in the DNA fragment distribution pattern.

scholarly journals The Drosophila melanogaster Ortholog of RFWD3 Functions Independently of RAD51 During DNA Repair

2020 ◽  
Vol 10 (3) ◽  
pp. 999-1004
Author(s):  
Juan Carvajal-Garcia ◽  
Evan R. Gales ◽  
Dale A. Ramsden ◽  
Jeff Sekelsky
Keyword(s):  
Dna Repair ◽  
Drosophila Melanogaster ◽  
Ring Finger ◽  
Double Strand Breaks ◽  
Specific Gene ◽  
Genetic Screens ◽  
Strand Breaks ◽  
Ancestral Function ◽  
Human Ortholog

Repair of damaged DNA is required for the viability of all organisms. Studies in Drosophila melanogaster, driven by the power of genetic screens, pioneered the discovery and characterization of many genes and pathways involved in DNA repair in animals. However, fewer than half of the alleles identified in these screens have been mapped to a specific gene, leaving a potential for new discoveries in this field. Here we show that the previously uncharacterized mutagen sensitive gene mus302 codes for the Drosophila melanogaster ortholog of the E3 ubiquitin ligase RING finger and WD domain protein 3 (RFWD3). In human cells, RFWD3 promotes ubiquitylation of RPA and RAD51 to facilitate repair of collapsed replication forks and double-strand breaks through homologous recombination. Despite the high similarity in sequence to the human ortholog, our evidence fails to support a role for Mus302 in the repair of these types of damage. Last, we observe that the N-terminal third of RFWD3 is only found in mammals, but not in other vertebrates or invertebrates. We propose that the new N-terminal sequence accounts for the acquisition of a new biological function in mammals that explains the functional differences between the human and the fly orthologs, and that Drosophila Mus302 may retain the ancestral function of the protein.

scholarly journals Endogenous Hot Spots ofDe NovoTelomere Addition in the Yeast Genome Contain Proximal Enhancers That Bind Cdc13

2016 ◽  
Vol 36 (12) ◽  
pp. 1750-1763 ◽  
Author(s):  
Udochukwu C. Obodo ◽  
Esther A. Epum ◽  
Margaret H. Platts ◽  
Jacob Seloff ◽  
Nicole A. Dahlson ◽  
...  
Keyword(s):  
Hot Spots ◽  
High Frequency ◽  
Genome Stability ◽  
De Novo ◽  
Dna Binding Protein ◽  
Double Strand Breaks ◽  
Yeast Genome ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Mutagenic Process

DNA double-strand breaks (DSBs) pose a threat to genome stability and are repaired through multiple mechanisms. Rarely, telomerase, the enzyme that maintains telomeres, acts upon a DSB in a mutagenic process termed telomere healing. The probability of telomere addition is increased at specific genomic sequences termed sites of repair-associated telomere addition (SiRTAs). By monitoring repair of an induced DSB, we show that SiRTAs on chromosomes V and IX share a bipartite structure in which a core sequence (Core) is directly targeted by telomerase, while a proximal sequence (Stim) enhances the probability ofde novotelomere formation. The Stim and Core sequences are sufficient to confer a high frequency of telomere addition to an ectopic site. Cdc13, a single-stranded DNA binding protein that recruits telomerase to endogenous telomeres, is known to stimulatede novotelomere addition when artificially recruited to an induced DSB. Here we show that the ability of the Stim sequence to enhancede novotelomere addition correlates with its ability to bind Cdc13, indicating that natural sites at which telomere addition occurs at high frequency require binding by Cdc13 to a sequence 20 to 100 bp internal from the site at which telomerase acts to initiatede novotelomere addition.

scholarly journals Homology Requirements for Targeting Heterologous Sequences During P-Induced Gap Repair in Drosophila melanogaster  1

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 689-699 ◽  
Author(s):  
Tammy Dray ◽  
Gregory B Gloor
Keyword(s):  
Drosophila Melanogaster ◽  
Point Mutations ◽  
Double Strand Breaks ◽  
Yellow Gene ◽  
P Element ◽  
White Gene ◽  
Base Pairs ◽  
Strand Breaks ◽  
Distal Point ◽  
White Locus

The effect of homology on gene targeting was studied in the context of P-element-induced double-strand breaks at the white locus of Drosophila melanogaster. Double-strand breaks were made by excision of P-whd, a P-element insertion in the white gene. A nested set of repair templates was generated that contained the 8 kilobase (kb) yellow gene embedded within varying amounts of white gene sequence. Repair with unlimited homology was also analyzed. Flies were scored phenotypically for conversion of the yellow gene to the white locus. Targeting of the yellow gene was abolished when all of the 3′ homology was removed. Increases in template homology up to 51 base pairs (bp) did not significantly promote targeting. Maximum conversion was observed with a construct containing 493 bp of homology, without a significant increase in frequency when homology extended to the tips of the chromosome. These results demonstrate that the homology requirements for targeting a large heterologous insertion are quite different than those for a point mutation. Furthermore, heterologous insertions strongly affect the homology requirements for the conversion of distal point mutations. Several aberrant conversion tracts, which arose from templates that contained reduced homology, also were examined and characterized.

Repair of deletions and double-strand gaps by homologous recombination in a mammalian in vitro system

1991 ◽  
Vol 11 (1) ◽  
pp. 445-457
Author(s):  
R Jessberger ◽  
P Berg
Keyword(s):  
Dna Sequences ◽  
Kanamycin Resistance ◽  
Double Strand Breaks ◽  
Strand Transfer ◽  
In Vitro System ◽  
Strand Breaks ◽  
Nuclear Extracts ◽  
Dna Strand

We have designed an in vitro system using mammalian nuclear extracts, or fractions derived from them, that can restore the sequences missing at double-strand breaks (gaps) or in deletions. The recombination substrates consist of (i) recipient DNA, pSV2neo with gaps or deletions ranging from 70 to 390 bp in the neo sequence, and (ii) donor DNAs with either complete homology to the recipient (pSV2neo) or plasmids whose homology with pSV2neo is limited to a 1.0- to 1.3-kbp neo segment spanning the gaps or deletions. Incubation of these substrates with various enzyme fractions results in repair of the recipient DNA's disrupted neo gene. The recombinational repair was monitored by transforming recA Escherichia coli to kanamycin resistance and by a new assay which measures the extent of DNA strand transfer from the donor substrate to the recipient DNA. Thus, either streptavidin- or antidigoxigenin-tagged beads are used to separate the biotinylated or digoxigeninylated recipient DNA, respectively, after incubation with the isotopically labeled donor DNA. In contrast to the transfection assay, the DNA strand transfer measurements are direct, quantitative, rapid, and easy, and they provide starting material for the characterization of the recombination products and intermediates. Accordingly, DNA bound to beads serves as a suitable template for the polymerase chain reaction. With appropriate pairs of oligonucleotide primers, we have confirmed that both gaps and deletions are fully repaired, that deletions can be transferred from the recipient DNA to the donor's intact neo sequence, and that cointegrant molecules containing donor and recipient DNA sequences are formed.

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