scholarly journals 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.

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.

The minimum amount of homology required for homologous recombination in mammalian cells

1984 ◽  
Vol 4 (11) ◽  
pp. 2253-2258
Author(s):  
J Rubnitz ◽  
S Subramani
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Recombination Frequency ◽  
Plaque Assay ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Minimum Amount ◽  
Base Pairs ◽  
Viral Plaque

Although DNA sequence homology is believed to be a prerequisite for homologous recombination events in procaryotes and eucaryotes, no systematic study has been done on the minimum amount of homology required for homologous recombination in mammalian cells. We have used simian virus 40-pBR322 hybrid plasmids constructed in vitro as substrates to quantitate intramolecular homologous recombination in cultured monkey cells. Excision of wild-type simian virus 40 DNA by homologous recombination was scored by the viral plaque assay. Using a series of plasmids containing 0 to 243 base pairs of homology, we have shown that the recombination frequency decreases as the homology is reduced, with the sharpest drop in recombination frequency occurring when the homology was reduced from 214 to 163 base pairs. However, low recombination frequencies were also observed with as little as 14 base pairs of homology.

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.

scholarly journals 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.

HOMOLOGOUS RECOMBINATION BETWEEN AUTONOMOUSLY REPLICATING PLASMIDS IN MAMMALIAN CELLS

Genetics ◽  
1985 ◽  
Vol 111 (2) ◽  
pp. 375-388
Author(s):  
David Ayares ◽  
James Spencer ◽  
Faina Schwartz ◽  
Brian Morse ◽  
Raju Kucherlapati
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Recombination Frequency ◽  
Shuttle Vector ◽  
Cell System ◽  
Double Strand Breaks ◽  
Low Molecular Weight ◽  
Strand Breaks ◽  
E Coli ◽  
Cos Cells

ABSTRACT The ability of autonomously replicating plasmids to recombine in mammalian cells was investigated. Two deletion plasmids of the eukaryotic-prokaryotic shuttle vector pSV2neo were cotransfected into transformed monkey COS cells. Examination of the low molecular weight DNA isolated after 48 hr of incubation revealed that recombination between the plasmids had occurred. The DNA was also used to transform recA- E. coli. Yield of neo R colonies signified homologous recombination. Examination of the plasmid DNA from these colonies confirmed this view. Double-strand breaks in one or both of the input plasmids at the sites of deletion resulted in an enhancement of recombination frequency. The recombination process yielded monomeric and dimeric molecules. Examination of these molecules revealed that reciprocal recombination as well as gene conversion events were involved in the generation of plasmids bearing an intact neo gene. The COS cell system we describe is analogous to study of bacteriophage recombination and yeast random-spore analysis.

scholarly journals The minimum amount of homology required for homologous recombination in mammalian cells.

1984 ◽  
Vol 4 (11) ◽  
pp. 2253-2258 ◽  
Author(s):  
J Rubnitz ◽  
S Subramani
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Recombination Frequency ◽  
Plaque Assay ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Minimum Amount ◽  
Base Pairs ◽  
Viral Plaque

Although DNA sequence homology is believed to be a prerequisite for homologous recombination events in procaryotes and eucaryotes, no systematic study has been done on the minimum amount of homology required for homologous recombination in mammalian cells. We have used simian virus 40-pBR322 hybrid plasmids constructed in vitro as substrates to quantitate intramolecular homologous recombination in cultured monkey cells. Excision of wild-type simian virus 40 DNA by homologous recombination was scored by the viral plaque assay. Using a series of plasmids containing 0 to 243 base pairs of homology, we have shown that the recombination frequency decreases as the homology is reduced, with the sharpest drop in recombination frequency occurring when the homology was reduced from 214 to 163 base pairs. However, low recombination frequencies were also observed with as little as 14 base pairs of homology.

scholarly journals Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells.

1997 ◽  
Vol 17 (11) ◽  
pp. 6386-6393 ◽  
Author(s):  
D G Taghian ◽  
J A Nickoloff
Keyword(s):  
Homologous Recombination ◽  
Gene Conversion ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Double Strand Breaks ◽  
Chromosomal Dna ◽  
Exogenous Dna ◽  
Strand Breaks ◽  
Conversion Tract

Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting. Transcription also stimulates spontaneous recombination by an unknown mechanism. We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA. One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo. The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms). This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels. Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1%. Strikingly, 97% of these products arose by gene conversion. Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal. DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing). In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over. For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription. Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion.

Double-strand-break-induced homologous recombination in mammalian cells

2001 ◽  
Vol 29 (2) ◽  
pp. 196-201 ◽  
Author(s):  
R. D. Johnson ◽  
M. Jasin
Keyword(s):  
Homologous Recombination ◽  
Sister Chromatid ◽  
Mammalian Cells ◽  
Indirect Evidence ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Crossing Over ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks

In mammalian cells, the repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. Indirect evidence, including that from gene targeting and random integration experiments, had suggested that non-homologous mechanisms were significantly more frequent than homologous ones. However, more recent experiments indicate that homologous recombination is also a prominent DSB repair pathway. These experiments show that mammalian cells use homologous sequences located at multiple positions throughout the genome to repair a DSB. However, template preference appears to be biased, with the sister chromatid being preferred by 2–3 orders of magnitude over a homologous or heterologous chromosome. The outcome of homologous recombination in mammalian cells is predominantly gene conversion that is not associated with crossing-over. The preference for the sister chromatid and the bias against crossing-over seen in mitotic mammalian cells may have developed in order to reduce the potential for genome alterations that could occur when other homologous repair templates are utilized. In attempts to understand further the mechanism of homologous recombination, the proteins that promote this process are beginning to be identified. To date, four mammalian proteins have been demonstrated conclusively to be involved in DSB repair by homologous recombination: Rad54, XRCC2, XRCC3 and BRCAI. This paper summarizes results from a number of recent studies.

scholarly journals Homologous recombination preferentially repairs heat-induced DNA double-strand breaks in mammalian cells

2016 ◽  
Vol 33 (3) ◽  
pp. 336-342 ◽  
Author(s):  
Akihisa Takahashi ◽  
Eiichiro Mori ◽  
Yosuke Nakagawa ◽  
Atsuhisa Kajihara ◽  
Tadaaki Kirita ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

scholarly journals Efficient In Vitro System of Homologous Recombination in Brome Mosaic Bromovirus

2006 ◽  
Vol 80 (12) ◽  
pp. 6182-6187 ◽  
Author(s):  
Rafal Wierzchoslawski ◽  
Jozef J. Bujarski
Keyword(s):  
Homologous Recombination ◽  
Recombination Frequency ◽  
In Vivo Studies ◽  
Rna Recombination ◽  
In Vitro System ◽  
Brome Mosaic Bromovirus ◽  
Subgenomic Promoter

ABSTRACT Recent in vivo studies have revealed that the subgenomic promoter (sgp) in brome mosaic bromovirus (BMV) RNA3 supports frequent homologous recombination events (R. Wierzchoslawski, A. Dzianott, and J. Bujarski, J. Virol. 78:8552-8564, 2004). In this paper, we describe an sgp-driven in vitro system that supports efficient RNA3 crossovers. A 1:1 mixture of two (−)-sense RNA3 templates was copied with either a BMV replicase (RdRp) preparation or recombinant BMV protein 2a. The BMV replicase enzyme supported a lower recombination frequency than 2a, demonstrating a role of other viral and/or host factors. The described in vitro system will allow us to study the mechanism of homologous RNA recombination.

Sign in / Sign up

Export Citation Format

Share Document