Characterization of nonconservative homologous junctions in mammalian cells.

Desautels, Lyne; Brouillette, S; Wallenburg, John C.; Belmaaza, Abdellah; Gusew, Nadine; Trudel, Pierre; Chartrand, Pascal

doi:10.1128/mcb.10.12.6613

Cited by 16 publications

(4 citation statements)

References 35 publications

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“…Resolution of the heteroduplexes in various ways via mismatch repair can then generate the observed recombination products. The mosaic pattern of parental markers detected in the recombinants is similar to other observations of nonconservative recombination products in mammalian cells (11). In particular, the predicted heteroduplex between the repeated supF sequences may contain several mismatches.…”

supporting

confidence: 66%

Recombination Induced by Triple-Helix-Targeted DNA Damage in Mammalian Cells

Faruqi

Seidman

Segal

et al. 1996

Molecular and Cellular Biology

105

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Gene therapy has been hindered by the low frequency of homologous recombination in mammalian cells. To stimulate recombination, we investigated the use of triple-helix-forming oligonucleotides (TFOs) to target DNA damage to a selected site within cells. By treating cells with TFOs linked to psoralen, recombination was induced within a simian virus 40 vector carrying two mutant copies of the supF tRNA reporter gene. Gene conversion events, as well as mutations at the target site, were also observed. The variety of products suggests that multiple cellular pathways can act on the targeted damage, and data showing that the triple helix can influence these pathways are presented. The ability to specifically induce recombination or gene conversion within mammalian cells by using TFOs may provide a new research tool and may eventually lead to novel applications in gene therapy.Gene targeting has been used to introduce foreign DNA into the genomes of mammalian cells through site-specific homologous recombination. By targeting specific genes in embryonic stem cells, it is now possible to study the role of individual genes in laboratory animals (27). However, the frequency of targeted recombination in mammalian cells is low, and the ratio of homologous to nonhomologous integration events is often unfavorable (15,44). This low frequency of homologous recombination limits the use of this technology for the purpose of gene therapy, and therefore efforts have been made to improve the efficiency of gene targeting.Previous studies have indicated that a double-strand break (DSB) within the region of homology in the donor molecule can enhance the recombination and targeting frequency (16,22). However, this increase is modest, and the DSB is likely to result in an increase in nonhomologous recombination as well. Recently, efforts have also been directed toward modification of the recipient site to create a substrate prone to homologous recombination. It has been shown that a site-specific endonuclease, I-SceI, can induce DSBs within extrachromosomal and genomic DNA designed to carry the rare 18-bp recognition site. This strategy has been used to induce intermolecular recombination in both Xenopus oocytes (40) and mammalian cells (5, 37). However, this strategy has limited practical application, as it involves the prior introduction of the recognition site within the genome.Besides DSBs, other types of DNA damage have been shown to be recombinogenic. These include DNA damage from UV radiation (45), chemical carcinogens (52), and photoreactive molecules such as psoralen (38). Psoralens are DNA-damaging agents that intercalate into DNA and form covalent monoadducts (MAs) and interstrand cross-links (XLs) upon exposure to near-UV light (UVA). Photo-induced psoralen interstrand XLs, and to a lesser extent MAs, can induce recombination in bacterial (7, 25), yeast (38), and mammalian (48) cells.In these studies of psoralen-induced recombination, however, there was no specificity to the distribution of psoralen adducts other than that der...

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supporting

confidence: 66%

Recombination Induced by Triple-Helix-Targeted DNA Damage in Mammalian Cells

Faruqi

Seidman

Segal

et al. 1996

Molecular and Cellular Biology

105

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“…Go/G1 arrested cells did not efficiently degrade injected DNA (Fig. 4B), suggesting that these cells have low levels of the exonuclease activities previously reported to be involved in repair and recombination events (43). On the other hand, little or no product was obtained when primers positioned on either side of the double strand break were used for amplification (Fig.…”

Section: Resultsmentioning

confidence: 90%

Sensitivity and selectivity of the DNA damage sensor responsible for activating p53-dependent G1 arrest.

Huang

Clarkin

Wahl

1996

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

342

176

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The tumor suppressor p53 contributes to maintaining genome stability by inducing a cell cycle arrest or apoptosis in response to conditions that generate DNA damage. Nuclear injection of linearized plasmid DNA, circular DNA with a large gap, or single-stranded circular phagemid is sufficient to induce a p53-dependent arrest. Supercoiled and nicked plasmid DNA, and circular DNA with a small gap were ineffective. Titration experiments indicate that the arrest mechanism in normal human fibroblasts can be activated by very few double strand breaks, and only one may be sufficient.Polymerase chain reaction assays showed that end-joining activity is low in serum-arrested human fibroblasts, and that higher joining activity occurs as cells proceed through G, or into S phase. We propose that the exquisite sensitivity of the p53-dependent G1 arrest is partly due to inefficient repair of certain types of DNA damage in early G1.Normal cells evolve into cancers by a process of clonal evolution involving the accumulation of multiple genetic alterations. Many of these changes are initiated by chromosome breakage. Through induction of apoptosis or cell cycle arrest, a p53-dependent mechanism effectively prevents DNA damage present in G, from being replicated in S phase (1-6). As DNA breakage is likely to be the first step in the process of generating gene amplification, translocations, and deletions, it is understandable why cells with an intact p53 pathway do not produce descendants with such alterations at experimentally measurable rates (7-9). In contrast, inactivation of p53 alone allows immortalized nontumorigenic cells and primary fibroblasts to cycle in the presence of chromosome breaks and to undergo gene amplification at high rates (4, 10, 11). It is not surprising, therefore, that loss of p53 function is highly selected during cancer progression, and that defects in the p53 gene occur in more than 50% of human cancers (12).The specific signals that induce p53-dependent G1 arrest remain to be elucidated. Previous studies showed that ultraviolet light, ionizing radiation, and a variety of chemotherapeutic agents increase p53 levels (3,4,13,14) and alter expression of p53 responsive genes (3,(15)(16)(17)

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“…There have been a few reports of patchy conversion tracts for recombination in yeast (16) and mammalian cells (7,13), although the latter two studies did not involve intrachromosomal events. A continuous conversion tract could be produced by a heteroduplex DNA (hDNA) intermediate (30) that is repaired as a continuous span by using a single template or remains unrepaired and is restored to homoduplex by replication.…”

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confidence: 99%

Fine-Resolution Analysis of Products of Intrachromosomal Homeologous Recombination in Mammalian Cells

Yang

Waldman

1997

Molecular and Cellular Biology

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Mouse Ltk؊ cell lines that contained a herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene with a 16-bp insertion mutation linked to either a defective HSV-2 tk gene or a hybrid tk sequence comprised of HSV-1 and HSV-2 tk sequences were constructed. HSV-1 and HSV-2 tk genes have 81% nucleotide identity and hence are homeologous. Correction of the insertion mutant HSV-1 tk gene via recombination with the hybrid tk sequence required an exchange between homeologous tk sequences, although recombination could initiate within a region of significant sequence identity. Seven cell lines containing linked HSV-1 and HSV-1-HSV-2 hybrid tk sequences gave rise to tk ؉ segregants at an average rate of 10 ؊8 events per cell division. DNA sequencing revealed that each recombinant from these lines displayed an apparent gene conversion which involved an accurate transfer of an uninterrupted block of information between homeologous tk sequences. Conversion tract lengths ranged from 35 to >330 bp. In contrast, cell lines containing linked HSV-1 and HSV-2 tk sequences with no significant stretches of sequence identity had an overall rate of homeologous recombination of <10 ؊9. One such cell line produced homeologous recombinants at a rate of 10 ؊8. Strikingly, all homeologous recombinants from this latter cell line were due to crossovers between the HSV-1 and HSV-2 tk genes. Our results, which provide the first detailed analysis of homeologous recombination within a mammalian genome, suggest that rearrangements in mammalian genomes are regulated by the degree of sequence divergence located at the site of recombination initiation.Recombination between similar but imperfectly matched sequences is referred to as homeologous recombination. Homeologous recombination events have been recovered in several organisms, including yeast (2,12,15,16,18,31,32,34,35,40,43), bacteria (28, 36-39, 44, 45), and mammalian cells (3,53). When homeologous recombination is detected, it is typically much less frequent than is recombination between highly homologous sequences. Low rates for homeologous recombination may help to conserve genomic integrity by preventing undesirable rearrangements. On the other hand, when homeologous recombination does occur, it can potentially lead to novel genes and chromosomes and may play an important role in the evolution of genomes. For these reasons, it is of considerable interest to gain knowledge about such events as they occur in a variety of organisms. We previously investigated intrachromosomal homeologous recombination between a defective herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) sequence and a closely linked HSV-2 tk sequence in the genome of mouse fibroblasts (52). HSV-1 and HSV-2 tk sequences have 81% sequence identity, with a 35-bp stretch of sequence identity being the longest (19, 49). The rate of intrachromosomal homeologous recombination between HSV-1 and HSV-2 tk sequences was reduced more than 1,000-fold compared with that of intrachromosomal recombination between nearly perfect...

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Characterization of nonconservative homologous junctions in mammalian cells.

Cited by 16 publications

References 35 publications

Recombination Induced by Triple-Helix-Targeted DNA Damage in Mammalian Cells

Recombination Induced by Triple-Helix-Targeted DNA Damage in Mammalian Cells

Sensitivity and selectivity of the DNA damage sensor responsible for activating p53-dependent G1 arrest.

Fine-Resolution Analysis of Products of Intrachromosomal Homeologous Recombination in Mammalian Cells

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