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.

scholarly journals Efficient gap repair in Drosophila melanogaster requires a maximum of 31 nucleotides of homologous sequence at the searching ends.

1997 ◽  
Vol 17 (2) ◽  
pp. 627-634 ◽  
Author(s):  
K J Keeler ◽  
G B Gloor
Keyword(s):  
Drosophila Melanogaster ◽  
Internal Conversion ◽  
Double Strand Breaks ◽  
P Element ◽  
White Gene ◽  
Homology Search ◽  
Homologous Sequence ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Gene Conversions

Double-strand breaks (DSB) were generated in the Drosophila melanogaster white gene by excision of the P-w(hd) element. An ectopic P-element vector carrying a modified white gene was used as a template for DSB repair. All template-dependent repair events were examined, and four different classes of events were recovered. The two most common products observed were gene conversions external to the P-w(hd) element and gene conversions (targeted transpositions) internal to the P-w(hd) element. These two events were equally frequent. Similar numbers for both orientations of internal conversion events were recovered. The results suggest that P-element excision occurs by a staggered cut that leaves behind at least 33 nucleotides of single-stranded sequence. Our results further demonstrate that an efficient homology search is conducted by the broken end with less than 31 nucleotides.

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.

How damaged is the biologically active subpopulation of transfected DNA?

1984 ◽  
Vol 4 (3) ◽  
pp. 387-398
Author(s):  
C T Wake ◽  
T Gudewicz ◽  
T Porter ◽  
A White ◽  
J H Wilson
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Double Strand Breaks ◽  
Biologically Active ◽  
Exact Nature ◽  
Dna Molecules ◽  
Base Pairs ◽  
Strand Breaks ◽  
Dna Ends ◽  
Prevalent Form

Relatively little is known about the damage suffered by transfected DNA molecules during their journey from outside the cell into the nucleus. To follow selectively the minor subpopulation that completes this journey, we devised a genetic approach using simian virus 40 DNA transfected with DEAE-dextran. We investigated this active subpopulation in three ways: (i) by assaying reciprocal pairs of mutant linear dimers which differed only in the arrangement of two mutant genomes; (ii) by assaying a series of wild-type oligomers which ranged from 1.1 to 2.0 simian virus 40 genomes in length; and (iii) by assaying linear monomers of simian virus 40 which were cleaved within a nonessential region to leave either sticky, blunt, or mismatched ends. We conclude from these studies that transfected DNA molecules in the active subpopulation are moderately damaged by fragmentation and modification of ends. As a whole, the active subpopulation suffers about one break per 5 to 15 kilobases, and about 15 to 20% of the molecules have one or both ends modified. Our analysis of fragmentation is consistent with the random introduction of double-strand breaks, whose cause and exact nature are unknown. Our analysis of end modification indicated that the most prevalent form of damage involved deletion or addition of less than 25 base pairs. In addition we demonstrated directly that the efficiencies of joining sticky, blunt, or mismatched ends are identical, verifying the apparent ability of cells to join nearly any two DNA ends and suggesting that the efficiency of joining approaches 100%. The design of these experiments ensured that the detected damage preceded viral replication and thus should be common to all DNAs transfected with DEAE-dextran and not specific for viral DNA. These measurements of damage within transfected DNA have important consequences for studies of homologous and nonhomologous recombination in somatic cells as is discussed.

scholarly journals TOPOVIBL-REC114 interaction regulates meiotic DNA double-strand breaks

2021 ◽  
Author(s):  
Alexandre Nore ◽  
Ariadna B Juarez-Martinez ◽  
Julie AJ Clement ◽  
Christine Brun ◽  
Bouboub Diagouraga ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Point Mutations ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Catalytic Complex ◽  
Strand Breaks ◽  
Genome Wide ◽  
Dna Cleavage Activity ◽  
Meiotic Dsbs

Meiosis requires the formation of programmed DNA double strand breaks (DSBs), essential for fertility and for generating genetic diversity. In male and female meiotic cells, DSBs are induced by the catalytic activity of the TOPOVIL complex formed by SPO11 and TOPOVIBL. To ensure genomic integrity, DNA cleavage activity is tightly regulated, and several accessory factors (REC114, MEI4, IHO1, and MEI1) are needed for DSB formation in mice. How and when these proteins act is not understood. Here, we show that REC114 is a direct partner of TOPOVIBL, and identified their conserved interacting domains by structural analysis. We then analysed the role of this interaction by monitoring meiotic DSBs in female and male mice carrying point mutations in TOPOVIBL that decrease or disrupt its binding to REC114. In these mutants, DSB activity was strongly reduced genome-wide in oocytes, but only in sub-telomeric regions in spermatocytes. In addition, in mutant spermatocytes, DSB activity was delayed in autosomes. These results provide evidence that REC114 is a key member of the TOPOVIL catalytic complex, and that the REC114/TOPOVIBL interaction ensures the efficiency and timing of DSB activity by integrating specific chromosomal features.

Timing of molecular events in meiosis in Saccharomyces cerevisiae: stable heteroduplex DNA is formed late in meiotic prophase

1993 ◽  
Vol 13 (1) ◽  
pp. 373-382 ◽  
Author(s):  
C Goyon ◽  
M Lichten
Keyword(s):  
Meiotic Prophase ◽  
Double Strand Breaks ◽  
Dna Breaks ◽  
Base Pairs ◽  
Heteroduplex Dna ◽  
Strand Breaks ◽  
Dna Structures ◽  
Double Strand Dna Breaks ◽  
Molecular Events ◽  
The Times

To better understand the means by which chromosomes pair and recombine during meiosis, we have determined the time of appearance of heteroduplex DNA relative to the times of appearance of double-strand DNA breaks and of mature recombined molecules. Site-specific double-strand breaks appeared early in meiosis and were formed and repaired with a timing consistent with a role for breaks as initiators of recombination. Heteroduplex-containing molecules appeared about 1 h after double-strand breaks and were followed shortly by crossover products and the first meiotic nuclear division. We conclude that parental chromosomes are stably joined in heteroduplex-containing structures late in meiotic prophase and that these structures are rapidly resolved to yield mature crossover products. If the chromosome pairing and synapsis observed earlier in meiotic prophase is mediated by formation of biparental DNA structures, these structures most likely either contain regions of non-Watson-Crick base pairs or contain regions of heteroduplex DNA that either are very short or dissociate during DNA purification. Two loci were examined in this study: the normal ARG4 locus, and an artificial locus consisting of an arg4-containing plasmid inserted at MAT. Remarkably, sequences in the ARG4 promoter that suffered double-strand cleavage at the normal ARG4 locus were not cut at significant levels when present at MAT::arg4. These results indicate that the formation of double-strand breaks during meiosis does not simply involve the specific recognition and cleavage of a short nucleotide sequence.

Effect of double-strand breaks on homologous recombination in mammalian cells and extracts

1985 ◽  
Vol 5 (12) ◽  
pp. 3331-3336
Author(s):  
K Y Song ◽  
L Chekuri ◽  
S Rauth ◽  
S Ehrlich ◽  
R Kucherlapati
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Recombination Frequency ◽  
Double Strand Breaks ◽  
Base Pairs ◽  
In Vitro System ◽  
Strand Breaks ◽  
Cell Extracts ◽  
Nuclear Extracts

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.

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 Indirect and direct evidence for DNA double–strand breaks in hypermutating immunoglobulin genes

2001 ◽  
Vol 356 (1405) ◽  
pp. 119-125 ◽  
Author(s):  
Heinz Jacobs ◽  
Klaus Rajewsky ◽  
Yosho Fukita ◽  
Linda Bross
Keyword(s):  
Somatic Hypermutation ◽  
Gene Segment ◽  
Point Mutations ◽  
Antigen Receptor ◽  
Double Strand Breaks ◽  
Mouse Strains ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Receptor Repertoire ◽  
Igh Locus

The generation of a diverse antigen receptor repertoire is fundamental for the functionality of the adaptive immune system. While the V(D)J recombination process that generates the primary antigen receptor repertoire is understood in great detail, it is still unclear by which mechanism immunoglobulin (Ig) genes are further diversified by somatic hypermutation. Using mouse strains that carry a non–functional, predefined V H D H J H gene segment in their IgH locus we demonstrate DNA double–strand breaks (DSBs) in and around V H D H J H in B cells undergoing somatic hypermutation. The generation of these DSBs depends on transcriptional activity, and their distribution along the V H D H J H segment parallels that of point mutations in the hypermutation domain. Furthermore, similar to hot spots of somatic hypermutation, 50–60% of all DSBs occur preferentially at RGYW motifs. DSBs may transiently dissociate the Ig promoter from the intronic enhancer to block further transcription and to initiate an error–prone nonhomologous DSB repair pathway. In accord with this model large deletions are frequently produced, along with point mutations, in a V H D H J H segment inserted together with its promoter into the IgH locus in inverted orientation. Our data suggest that DSBs are reaction intermediates of the mechanism underlying somatic hypermutation.

scholarly journals Sequences involved in temperature and ecdysterone-induced transcription are located in separate regions of a Drosophila melanogaster heat shock gene.

1986 ◽  
Vol 6 (2) ◽  
pp. 663-673 ◽  
Author(s):  
E Hoffman ◽  
V Corces
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Dna Sequences ◽  
Germ Line ◽  
Initiation Site ◽  
P Element ◽  
Heat Shock Gene ◽  
Base Pairs ◽  
Consensus Sequences ◽  
Shock Gene

The transcriptional regulation of the Drosophila melanogaster hsp27 (also called hsp28) gene was studied by introducing altered genes into the germ line by P element-mediated transformation. DNA sequences upstream of the gene were defined with respect to their effect on steroid hormone-induced and heat-induced transcription. These two types of control were found to be separable; the sequences responsible for 80% of heat-induced expression were located more than 1.1 kilobases upstream of the RNA initiation site, while the sequences responsible for the majority of ecdysterone induction were positioned downstream of the site at -227 base pairs. We have determined the DNA sequence of the intergenic region separating hsp23 and hsp27 and have located putative heat shock and ecdysterone consensus sequences. Our results indicate that the heat shock promoter of the hsp27 gene is organized quite differently from that of hsp70.

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