Spontaneous damage to DNA is frequent and may lead to cell death, cell senescence, or mutations. DNA double-strand breaks (DSBs) are of special interest because they are highly toxic and have been implicated in neurodegeneration, cancer, and aging. Until now, there has not been a reliable system allowing tunable induction of random DSBs without affecting other macromolecules or cell functions. Here, we describe an adenoviral-based, doxycycline-mediated, and tamoxifen-dependent system for quantitative introduction of DSBs in mammalian cells. We generated a single adenoviral vector containing a tet-inducible, composite SacI restriction endonuclease/estrogen receptor (ERT2) gene, and a constitutively expressed reverse transactivator (rtTA) gene. Transduced mouse embryonic fibroblasts—as well as mouse liver cells in vivo—demonstrated a high level of DSBs in response to treatment with doxycycline and tamoxifen. We show that the amount of induced DSBs can be titrated by doxycycline dose and duration of treatment. This system should be useful for studying the processing of randomly induced DSBs and their effects on cell fate, without the side effects normally associated with radiation or chemical treatment.

Introduction

Physical and chemical methods commonly used for generating DNA double-strand breaks (DSBs) in mammalian cells induce a spectrum of lesions and can affect other biological macromolecules as well as normal cell physiology. An alternative way of inducing DSBs in living cells is through ectopic expression of proteins possessing an endonuclease activity. The endonucleases recognize specific sequences, directly cleave DNA and, unlike physical and chemical methods, generate no other lesions and have no adverse physiological effects. Rare-cutting homing endonucleases I-SceI (1), I-CreI, and I-PpoI (2,3) have been successfully used for this purpose. The size of recognition sites for homing endonucleases (18, 24, and 15 bp, respectively) determines their low frequency in the mammalian genome and makes them ideal tools for studying DSB repair. However, the same feature makes these enzymes less useful for studying stochastically distributed DSBs (as in the natural situation) and the associated processing events and various cellular end points. This limitation can be overcome by the use of bacterial restriction endonucleases (REs). REs generally have shorter recognition sites (typically 4–8 bp) and are present in the genome at a much higher frequency. REs were shown to cause DSBs and induce chromosomal aberrations when directly electroporated or genetically expressed in eukaryotic cells (4,5,6,7). However, the utility of these approaches is seriously compromised by the necessity of special cell treatment to permit access to protein or plasmid DNA, and by the lack of RE activity regulation.

Here we describe an adenoviral-based, doxycycline-mediated, and tamoxifen-dependent system for the quantitative introduction of DSBs into genomic DNA. The generated construct contains both parts of the tet-inducible system: a tet-inducible promoter driving expression of SacI RE fused to mutated estrogen receptor gene ERT2 (8), and reverse transactivator (rtTA) driven by the CMV promoter in the backbone of an adenoviral vector. The SacI RE has a 6-bp recognition site (GAGCTC) and creates cohesive ends with a four-nucleotide overhang after cleaving DNA. Cultured mouse embryonic fibroblasts transduced with this virus, as well as mouse liver cells in vivo after tail vein injection of the virus suspension, demonstrate inducible expression of SacI in response to doxycycline treatment, which results in the increased expression of a DSB marker. We show that the level of DNA damage can be controlled by the dose of doxycycline and duration of drug application.

Materials and methods

Vector construction and virus production

SacIR coding sequence lacking the stop codon was amplified from genomic DNA of Streptomyces achromogenes (provided by New England BioLabs, Ipswich, MA, USA) with Pfx polymerase (Invitrogen, Carlsbad, CA, USA) and cloned in frame with a V5 epitope into an expression vector containing the CMV promoter to get pCMV-SacIV5. Primers used were 5′-CACCATGGGAATAACAATTAAAAAGAGCACGGCG-3′ (forward) and 5′-CGTTTCAGGGAAGATCTCAGCCCA-3′ (reverse). The SacI coding sequence was fused with the mutated estrogen receptor gene ERT2, excised from pCre-ERT2 plasmid (provided by P. Chambon, Illkirch, France) resulting in a tamoxifen-inducible variant of SacI. The SacI-ERT2 coding sequence was cloned together with V5 epitope into a pTRE-Tight vector (Clontech, Mountain View, CA, USA) to get pTight-SEV5.

Tight-SEV5-pA and CMV-rtTA-pA expression cassettes were PCR-amplified with Pfx polymerase (Invitrogen) from pTight-SEV5 and pTet-ON (Clontech), respectively, and cloned into pBluescript KS+ vector in head-to-tail orientation. Tight-SEV5-pA-CMV-rtTA-pA fragment was PCR-amplified with High Fidelity Platinum Taq Polymerase (Invitrogen) and sub-cloned into an entry vector using a pCR8/GW/TOPO TA Cloning Kit (Invitrogen). The resulting pGW/Tight-SEV5-CMV-rtTA entry vector was used to clone the Tight-SEV5-pA-CMV-rtTA-pA cassette into a pAd/PL-DEST vector (ViraPower Adenoviral Expression System Kit, Invitrogen) to get pAd/TSCR. All cloning work with plasmids containing the SacIR gene was performed in Stbl3 Escherichia coli strain (Invitrogen) transformed with a plasmid that expresses SacI methylase (provided by New England BioLabs).

Adenoviral stocks (A/TSCR) were produced in 293A/tTS cells according to the manufacturer's instructions and titrated with Adenoviral Rapid Titer Kit (Clontech).

Western blotting

Protein extraction from cultured cells was performed with a Whole Cell Extraction Kit (Chemicon, Billerica, MA, USA) after separation on NuPAGE 4–12% Bis-Tris Gel (Invitrogen) in MOPS SDS running buffer (Invitrogen). Proteins were transferred on PVDF membrane using an iBlot Gel Transfer System (Invitrogen). The primary antibodies used were monoclonal anti-V5 (1:3000; Invitrogen), monoclonal anti-GAPDH (1:100000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and monoclonal anti-phospho-histone H2AX (1:3000; Upstate, Billerica, MA, USA). The secondary antibodies used were HRP-conjugated goat anti-mouse (1:5000; Santa Cruz Biotechnology). HRP was detected with Immun-Star HRP kit (Bio-Rad Laboratories, Hercules, CA, USA).