Homologous Recombination between Repeated Chromosomal Sequences in Mouse Cells

Liskay, R. Michael; Stachelek, J. L.; Letsou, Anthea

doi:10.1101/sqb.1984.049.01.021

Cited by 112 publications

(87 citation statements)

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“…The average gene conversion frequency (Table 3) was approximately fivefold higher than the average single-crossover frequency we have measured earlier (20). This result is consistent with those of Liskay et al (10,11), who found that gene conversion between defective tk genes occurred more frequently than reciprocal recombination. The gene conversion frequencies in Table 3 were 16-to 67-fold higher than the background reversion frequency of the neoX gene.…”

Section: Discussionsupporting

confidence: 82%

“…Thus, it has been difficult to assign frequencies to each type of event independently without doing fluctuation tests to estimate frequencies and characterizing sufficiently large numbers of independently derived recombination products from different parental cell lines to determine the relative proportions of reciprocal and nonreciprocal recombination. With these caveats in mind, the impression one gets from the published literature is that in some cell lines (11,16) gene conversion occurs 80% of the time, while reciprocal recombination occurs 20% of the time, whereas in others, reciprocal recombination predominates over nonreciprocal recombination (16).…”

mentioning

confidence: 98%

“…Recent experiments on chromosomal homologous recombination in a number of laboratories have relied on the use of either overlapping truncated fragments or mutant copies of the herpesvirus thymidine kinase (tk) (9)(10)(11) or neomycin resistance (neo) gene (16,20) to detect recombination events which restore one or more copies of the selected marker gene. Although these studies have measured recombination frequencies and characterized various reciprocal and nonreciprocal intrachromosomal recombination events, attempts to estimate the true frequencies of these events have been complicated by the ability of the recombination substrates to undergo both reciprocal and nonreciprocal recombination.…”

mentioning

confidence: 99%

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Extrachromosomal and chromosomal gene conversion in mammalian cells.

Rubnitz¹,

Subramani²

1986

Mol. Cell. Biol.

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We constructed substrates to study gene conversion in mammalian cells specifically without the complication of reciprocal recombination events. These substrates contain both an insertion mutation of the neomycin resistance gene (neoX) and an internal, homologous fragment of the neo gene (neo-526), such that gene conversion from neo-526 to neoX restores a functional neo gene. Although two reciprocal recombination events can also produce an intact neo gene, these double recombination events occur much less frequently that gene conversion in mammalian cells, We used our substrates to characterize extrachromosomal gene conversion in recombination-deficient bacteria and in monkey COS cells. Chromosomal recombination was also studied after stable integration of these substrates into the genome of mouse 3T6 cells. All extrachromosomal and chromosomal recombination events analyzed in mammalian cells resulted from gene conversion. Chromosomal gene conversion events occurred at frequencies of about 10(-6) per cell generation and restored a functional neo gene without overall effects on sequence organization.

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Section: Discussionsupporting

confidence: 82%

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confidence: 98%

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confidence: 99%

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Extrachromosomal and chromosomal gene conversion in mammalian cells.

Rubnitz¹,

Subramani²

1986

Mol. Cell. Biol.

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“…The questions of how long the stretch of homology must be and how closely the sequences must match within this stretch have been approached with various experimental systems in bacteria (2)(3)(4)(5), yeast (6)(7)(8), and mammals (9)(10)(11)(12)(13)(14)(15). The general conclusion from these studies is that the recombination mechanisms are much more sensitive to degree of homology than would be predicted purely on the basis of DNA-duplex stability.…”

Section: Introductionmentioning

confidence: 87%

DNA homology requirements for mitotic gap repair in Drosophila.

Nassif

Engels

1993

Proc. Natl. Acad. Sci. U.S.A.

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We used P transposable-element mobilization to study the repair of double-strand DNA breaks in Drosophila melanogaster premeiotic germ cells. Distribution of conversion tracts was found to be largely unaffected by changes in the length of sequence homology between the broken ends and the template, suggesting that only a short match is required. However, the frequency of repair was highly sensitive to single-base mismatches within the homologous region, ranging from 19% reversion when there were no mismatches to 5% when 15 mismatches were present over a 3455-bp span.Efficient recombination between two stretches of DNA requires that the two sequences locate each other in the genome and form the stable recombinational intermediate complex. Some degree of homology between the two interacting sequences is needed for efficient recombination by most pathways (1).The questions of how long the stretch of homology must be and how closely the sequences must match within this stretch have been approached with various experimental systems in bacteria (2-5), yeast (6-8), and mammals (9-15). The general conclusion from these studies is that the recombination mechanisms are much more sensitive to degree of homology than would be predicted purely on the basis of DNA-duplex stability. In Escherichia coli, for example, a single-base mismatch in 53 bp resulted in a 4-fold reduction in recombination (4), and a 40-fold reduction was seen when the mismatch proportion was increased to 10% (3). The minimum length of homology required has been estimated at 20-50 bp for E. coli (2-4) and 63-89 bp for Saccharomyces cerevisiae (7). For mammalian cells, estimates range from 163 to 300 bp (9, 13), but some studies indicate efficient recombination with as little as 20 bp (9, 10). One suggestion (2, 5) is that the length and stringency of homology requirements reflect an evolutionary adaptation to prevent recombination between unrelated sites and are, thus, expected to depend on size and complexity of the genome.In this report we make use of the recently developed method of transposable-element-induced gap repair to address the question of homology requirements for recombinational DNA repair. P elements are DNA transposons that have been used for a variety oftechniques in Drosophila (16). Recent data (17-19) led to a model in which P element excision leaves behind a double-stranded DNA break. According to this interpretation, such breaks are often extended into various-sized gaps through exonuclease activity. Repair occurs when the ends of the gap invade a homologous DNA sequence from which to copy the missing genetic information. The result is unidirectional transfer of information from the template sequence to the excision site. The template can be the sister strand or an ectopic site located elsewhere in the genome or extrachromosomally (17,18,20).The system allows us to examine the length and stringency of homology requirements in Drosophila melanogaster. A further advantage of this approach is that recombination occurs between sequ...

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“…All plasmids are based on the vector pJS-1, which is identical to pSV2neo (Southern and Berg 1982) except for several restriction site modifications previously described (Liskay et al 1984). Each recombination substrate contains a herpes simplex type 1 (HSV-1) thymidine kinase (tk) gene rendered nonfunctional by insertion of an 8-bp XhoI linker after nucleotide position 1215 or after nucleotide position 1035 of the tk gene [nucleotide numbering according to Wagner et al (1981)].…”

Section: Methodsmentioning

confidence: 99%

Genetic Exchange Between Homeologous Sequences in Mammalian Chromosomes Is Averted by Local Homology Requirements for Initiation and Resolution of Recombination

et al. 2006

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We examined the mechanism by which recombination between imperfectly matched sequences (homeologous recombination) is suppressed in mammalian chromosomes. DNA substrates were constructed, each containing a thymidine kinase (tk) gene disrupted by insertion of an XhoI linker and referred to as a ''recipient'' gene. Each substrate also contained one of several ''donor'' tk sequences that could potentially correct the recipient gene via recombination. Each donor sequence either was perfectly homologous to the recipient gene or contained homeologous sequence sharing only 80% identity with the recipient gene. Mouse Ltk À fibroblasts were stably transfected with the various substrates and tk 1 segregants produced via intrachromosomal recombination were recovered. We observed exclusion of homeologous sequence from gene conversion tracts when homeologous sequence was positioned adjacent to homologous sequence in the donor but not when homeologous sequence was surrounded by homology in the donor. Our results support a model in which homeologous recombination in mammalian chromosomes is suppressed by a nondestructive dismantling of mismatched heteroduplex DNA (hDNA) intermediates. We suggest that mammalian cells do not dismantle mismatched hDNA by responding to mismatches in hDNA per se but rather rejection of mismatched hDNA appears to be driven by a requirement for localized homology for resolution of recombination.

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Homologous Recombination between Repeated Chromosomal Sequences in Mouse Cells

Cited by 112 publications

References 0 publications

Extrachromosomal and chromosomal gene conversion in mammalian cells.

Extrachromosomal and chromosomal gene conversion in mammalian cells.

DNA homology requirements for mitotic gap repair in Drosophila.

Genetic Exchange Between Homeologous Sequences in Mammalian Chromosomes Is Averted by Local Homology Requirements for Initiation and Resolution of Recombination

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