Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers. The use of rare cutting DNA endonucleases-such as homing endonucleases, also known as meganucleases-constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules-Amel3-Amel4 and Ini3-Ini4-cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo. Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo.
The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤0.1% to ∼6%) with that of homologous gene targeting (≤0.1% to ∼15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.
In order to unravel the mechanism that regulates transcription of protein-coding genes, we investigated the function of the p44 subunit of TFIIH, a basal transcription factor that is also involved in DNA repair. We have shown previously that mutations in the C terminus of the XPD helicase, another subunit of TFIIH, prevent its regulation by p44 (Coin, F., Bergmann, E., TremeauBravard, A., and Egly, J. M. (1999) EMBO 18, 1357-1366). By using a site-directed mutagenesis approach within the p44 region from amino acids 66 to 200, we indicate how a decrease in the interaction between p44 and XPD results in a decrease of the XPD helicase activity and leads to a defect in the first steps of the transcription reaction, namely the first phosphodiester bond formation and promoter clearance. We thus provide some explanation for the transcriptional defect found in SSL1 mutated yeast (Wang, Z., Buratowski, S., Svejstrup, J. Q., Feaver, W. J., Wu, X., Kornberg, R. D., Donahue, T. F., and Friedberg, E. C. (1995) Mol. Cell. Biol. 15, 2288 -2293). Moreover, this study shows how the activity of the the cyclin-dependent kinase-activating kinase associated with TFIIH complex in stimulating transcription is mediated in part by p44/XPD interaction.TFIIH is a multisubunit complex involved in two major DNA metabolism pathways, transcription and nucleotide excision repair (1). The transcriptionally active form of TFIIH, also called holo-TFIIH, includes core TFIIH, a five-subunit subcomplex constituted of XPB, p62, p52, p44, and p34, as well as XPD and the three subunits of the cyclin-dependent kinase (cdk) 1 -activating kinase complex (known as CAK): cdk7, cyclin H, and MAT1. Mutations in XPB and XPD genes, encoding the two DNA-dependent ATPase-associated helicases (2-7), are responsible for several rare autosomal recessive human genetic disorders, including xeroderma pigmentosum (XP), Cockayne syndrome, and trichothiodystrophy (8 -11).Investigations on cells derived from XP-B and XP-D patients showed a significant decrease of both transcription and NER activities (12, 13). Many XP-B and XP-D patients suffer from a high UV sensitivity due to the inability of their NER machinery to remove DNA damage (14, 15). The NER reaction is dependent on both helicases, XPB and XPD, that play an essential role in the formation of an open complex structure necessary for the subsequent incisions on each side of the lesion by the sitespecific endonucleases, ERCC1-XPF and XPG (1, 16 -18).In transcription, TFIIH is involved at different levels of the initiation step. A mutation in the ATP binding domain of XPB gives rise to a transcription defect due to an impaired promoter opening thus preventing the synthesis of the first phosphodiester bond by RNA polymerase II (RNA pol II) (19 -22). The role of the XPD helicase, although rather elusive, appears to be less essential for the transcription initiation process. The XPD helicase activity is not required for both promoter opening and first phosphodiester bond synthesis; nevertheless, it significantly stimula...
We demonstrate for the first time that meganucleases can be successfully delivered in animal and induce targeted genomic recombination in mice liver in toto. These results are an essential step towards the use of designed meganucleases and show the high potential of this technology in the field of gene therapy.
The p44 subunit plays a crucial role in the overall activity of the transcription/DNA repair factor TFIIH: on the one hand its N-terminal domain interacts with and regulates the XPD helicase (1, 2); on the other hand, as shown in the present study, it participates with the promoter escape reaction. Mutagenesis along with recombinant technology using the baculovirus/insect cells expression system allowed us to define the function of the two structural motifs of the C-terminal moiety of p44: mutations within the C4 zinc finger motif (residues 291-308) prevent incorporation of the p62 subunit within the core TFIIH. Double mutations in the RING finger motif (residues 345-385) allow the synthesis of the first phosphodiester bond by RNA polymerase II, but prevent its escape from the promoter. This highlights the role of transcription factor IIH in the various steps of the transcription initiation process.Accurate transcription of class II genes requires formation of a preinitiation complex composed of RNA polymerase II (RNA pol 1 II) and several general transcription factors including TFIIH (3). TFIIH, also involved in DNA repair and cell cycle control (4), plays a central role in the initiation of transcription due to its numerous enzymatic activities: the 3Ј-5Ј XPB helicase opens DNA around the promoter (5-7), whereas the 5Ј-3Ј XPD helicase is more likely devoted to the opening of DNA around a damage (8,9). In transcription the role of XPD seems to be more structural, since it allows the anchoring of CAK (cdk-activating kinase) complex to the core TFIIH, for optimal phosphorylation of RNA pol II (10), and nuclear receptors (11)(12)(13). p62, p52, and p34 have no defined functions, whereas p44 regulates the XPD helicase activity within TFIIH (1, 2). Mutations in the two helicases XPB and XPD are associated with three rare genetics disorders: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy (14,15). To understand the role of TFIIH in transcription, we have engaged projects on each subunit within TFIIH.Mutations in SSL1 (the yeast counterpart of p44) affect the genome stability of the yeast strain (16). Temperature-sensitive ssl1 yeast mutants are UV light-sensitive and defective in transcription, DNA repair, and likely in translation (17)(18)(19). These various defects highlight the central role of p44 within TFIIH, with the understanding that this subunit also interacts with almost all the other subunits of the core TFIIH both in human and in yeast (20,21).2 Having depicted the role of the N-terminal part of p44 which regulates the XPD helicase activity, we here focus our attention on the C terminus end. In the present study, we demonstrate the importance of the C-terminal cysteine-rich motif in preserving TFIIH architecture. We also show that double mutations in the RING finger motif affect the transcriptional activity of TFIIH by preventing its escape from the promoter. EXPERIMENTAL PROCEDURES Construction of Recombinant BaculovirusesExpressing TFIIH Subunits-Baculoviruses expressing the TFIIH subu...
Targeting DNA double-strand breaks is a powerful strategy for gene inactivation applications. Without the use of a repair plasmid, targeted mutagenesis can be achieved through Non-Homologous End joining (NHEJ) pathways. However, many of the DNA breaks produced by engineered nucleases may be subject to precise re-ligation without loss of genetic information and thus are likely to be unproductive. In this study, we combined engineered endonucleases and DNA-end processing enzymes to increase the efficiency of targeted mutagenesis, providing a robust and efficient method to (i) greatly improve targeted mutagenesis frequency up to 30-fold, and; (ii) control the nature of mutagenic events using meganucleases in conjunction with DNA-end processing enzymes in human primary cells.
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