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. 2008 Jan;28(1):154-64.
doi: 10.1128/MCB.00816-07. Epub 2007 Oct 29.

Transcription-associated recombination is dependent on replication in Mammalian cells

Ponnari Gottipati  1 , Tobias N Cassel, Linda Savolainen, Thomas Helleday
Affiliations

Transcription-associated recombination is dependent on replication in Mammalian cells

Ponnari Gottipati et al. Mol Cell Biol. 2008 Jan.

Abstract

Transcription can enhance recombination; this is a ubiquitous phenomenon from prokaryotes to higher eukaryotes. However, the mechanism of transcription-associated recombination in mammalian cells is poorly understood. Here we have developed a construct with a recombination substrate in which levels of recombination can be studied in the presence or absence of transcription. We observed a direct enhancement in recombination when transcription levels through the substrate were increased. This increase in homologous recombination following transcription is locus specific, since homologous recombination at the unrelated hprt gene is unaffected. In addition, we have shown that transcription-associated recombination involves both short-tract and long-tract gene conversions in mammalian cells, which are different from double-strand-break-induced recombination events caused by endonucleases. Transcription fails to enhance recombination in cells that are not in the S phase of the cell cycle. Furthermore, inhibition of transcription suppresses induction of recombination at stalled replication forks, suggesting that recombination may be involved in bypassing transcription during replication.

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Figures

FIG. 1.
FIG. 1.
Schematic illustration of the TARneo substrate. (A) The substrate contains two inactive neo repeats. The downstream neo gene is controlled by a constitutive thymidine kinase promoter and has a 3′ deletion, resulting in a nonfunctional gene product. The upstream neo gene includes the full coding sequence but has an I-SceI restriction site introduced that creates a stop codon. The two neo genes are in direct orientation, separated by a functional hygromycin resistance cassette. The full recombination substrate was subcloned into a commercial vector containing a bidirectional promoter (Pbi-1) based on a tetracycline-controlled system (TetOff) and allowed inducible transcription over the upstream neomycin gene. The TetOff promoter codirectionally controls the transcription of the construct and the reporter luciferase gene, enabling the quantification of transcription through the substrate (4). The Pbi-1 promoter is negatively controlled by the presence of the tetracycline derivative doxycycline (DOX). Transcription is turned off in the presence of doxycycline and turned on in its absence. The construct can revert back to its active form by recombination, and the recombinants can be selected using G-418. STGC gives rise to a recombination product in which the upstream neo gene controlled by the inducible promoter is reverted. The second recombination product is a result of either LTGC or SCE, in which the downstream neo gene is reverted. The primers used to distinguish between the products of STGC (Pf and Pr2) and LTGC/SCE (Pf and Pr1) are also depicted. (B) Presence of doxycycline during selection with G-418 turns off Pbi-1, leading to the selection of LTGC/SCE alone since the product of STGC is not expressed. (C) When there is no doxycycline during selection, both the products of STGC and LTGC/SCE are selected with G-418.
FIG. 2.
FIG. 2.
Locus-specific homologous recombination is enhanced by transcription, and transcription does not further enhance DSB-induced recombination. (A) Luciferase assay on the substrate in S8TofZM3 and S8TofZM24 cells confirms that 1 μg/ml of doxycycline effectively inhibits transcription in the TARneo substrate. (B) Recombination frequencies in the TARneo substrate in the S8TofZM3 and S8TofZM24 cells in the presence (transcription off) or absence (transcription on) of 1 μg/ml doxycycline. (C) Recombination frequencies in the TARneo and hprt substrates of S8TofZM3 cells in the presence or absence of 1 μg/ml doxycycline (transcription off or on, respectively). (D) Recombination frequencies in S8TofZM3 cells with or without pCMVI-SceI (with or without DSB) in the presence (transcription off) or absence (transcription on) of 1 μg/ml doxycycline. Results presented represent averages from at least three independent experiments, and error bars denote standard deviations. DOX, doxycycline; RLUs, relative light units.
FIG. 3.
FIG. 3.
Transcription enhances both short-tract and long-tract gene conversion events in mammalian cells. Recombination frequency (A) and Recombination rate (B) in the TARneo substrate of S8TofZM3 cells in the presence or absence of 1 μg/ml doxycycline to turn off or turn on transcription, respectively. Products of LTGC/SCE alone or both LTGC/SCE and STGC are distinguished by selection in the presence or absence of 1 μg/ml doxycycline, respectively. Results presented represent averages from at least three independent experiments, and error bars denote standard deviations. (C) PCR analysis of two colonies each from selection plates with or without doxycycline, expanded from the recombination assay (see panel A) in order to distinguish between the products of STGC (PCR product corresponds to the recombined upstream neomycin without the I-SceI site) and LTGC/SCE (PCR product corresponds to the upstream neomycin with the I-SceI site). The different types of recombination events detected genetically correspond to the expected molecular structure. The primers used are depicted in Fig. 1. R1, Pf plus Pr1; R2, Pf plus Pr2; β, β-actin primers; DOX, doxycycline.
FIG. 4.
FIG. 4.
Inhibition of transcription elongation does not further affect transcription-associated recombination. Luciferase activity of the TARneo substrate of S8TofZM3 cells after treatment with increasing doses of actinomycin D (A) or DRB (B). (C) Recombination frequency in the TARneo substrate of S8TofZM3 cells after treatment with transcription elongation inhibitors 1 nM actinomycin D and 75 μM DRB in the presence (no transcription) or absence (transcription) of 1 μg/ml doxycycline. Results presented represent average from at least three independent experiments, and error bars denote standard deviations. Dox, doxycycline; RLUs, relative light units.
FIG. 5.
FIG. 5.
Transcription-associated recombination in the TARneo substrate is S phase dependent. (A) Fluorescence-activated cell sorting data representing the cell cycle profiles of S8TofZM3 cells after treatment with 40 ng/ml nocodazole and 1 μg/ml doxycycline. The results presented are a representative of at least three independent experiments. (B) Luciferase activity of the S8TofZM3 cells during treatment with 40 ng/ml nocodazole and 1 μg/ml doxycycline. (C) Recombination frequencies of the TARneo substrate in S8TofZM3 cells after treatment with 40 ng/ml nocodazole in the presence or absence of 1 μg/ml doxycycline. (D) Recombination frequencies of the TARneo substrate in S8TofZM3 cells after treatment with 10 mM thymidine in the presence or absence of 1 μg/ml doxycycline. Results presented represent averages from at least three independent experiments, and error bars denote standard deviations. DOX, doxycycline; RLUs, relative light units.
FIG. 6.
FIG. 6.
Transcription-associated recombination is likely to be due to the collision between transcription and replication machineries. (A) Recombination frequency in the hprt substrate of SPD8 cells after treatment with 10 mM thymidine alone, the transcription elongation inhibitor DRB (75 μM) or actinomycin D (1 nM) alone, or both thymidine and transcription elongation inhibitors together. Results presented represent averages from at least three independent experiments, and error bars denote standard deviations. (B) Clonogenic survival of SPD8 cells after treatment with 10 mM thymidine, DRB (75 μM), or actinomycin D (1 nM) or both thymidine and transcription elongation inhibitors together. Results presented represent averages from at least three independent experiments, and error bars denote standard deviations. (C) Cell cycle profiles of SPD8 cells after treatment with 10 mM thymidine, DRB (75 μM), or actinomycin D (1 nM) or both thymidine and transcription elongation inhibitors together. The results presented here are representative of at least three independent experiments.
FIG. 7.
FIG. 7.
Transcription-associated recombination is independent of DNA damage signaling pathways. Recombination frequencies in the TARneo substrate of S8TofZM3 cells after treatment with the following PI 3-kinase-like kinase inhibitors: 10 μM of KU55933 (ATMi), 10 μM of KU51777 (DNA-PKi), 500 nM of CEP3891 (Chk1i), or 10 mM of caffeine (nonspecific inhibitor of PI 3-kinase-like kinases). Results presented represent averages from at least three independent experiments, and error bars denote standard deviations. DOX, doxycycline; RLUs, relative light units; ATMi, ATM inhibitor; DNA-PKi, DNA-PK inhibitor; Chk1i, Chk1 inhibitor.
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References

    1. Abraham, R. T. 2001. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev. 152177-2196. - PubMed
    1. Aguilera, A. 2002. The connection between transcription and genomic instability. EMBO J. 21195-201. - PMC - PubMed
    1. Arnaudeau, C., C. Lundin, and T. Helleday. 2001. DNA double-strand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells. J. Mol. Biol. 3071235-1245. - PubMed
    1. Baron, U., S. Freundlieb, M. Gossen, and H. Bujard. 1995. Co-regulation of two gene activities by tetracycline via a bidirectional promoter. Nucleic Acids Res. 233605-3606. - PMC - PubMed
    1. Bolderson, E., J. Scorah, T. Helleday, C. Smythe, and M. Meuth. 2004. ATM is required for the cellular response to thymidine induced replication fork stress. Hum. Mol. Genet. 132937-2945. - PubMed

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