Integration Specificity of Phage ϕC31 Integrase in the Human Genome

Chalberg, Thomas W.; Portlock, Joylette L.; Olivares, Eric C.; Thyagarajan, Bhaskar; Kirby, Patrick J.; Hillman, R. Tyler; Hoelters, Juergen; Calos, Michèle P.

doi:10.1016/j.jmb.2005.11.098

Cited by 228 publications

(299 citation statements)

References 55 publications

(84 reference statements)

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“…The 31 isolates mapped to 18 different loci that were distributed over 15 different chromosomes. Four sites were previously reported to be insertion hotspots of ϕC31-mediated integration (27), and 11 were new sites. A major locus responsible for 35% (11/31) of the isolates is a known ϕC31-integration hotspot in chromosome 19 q13.31, where an intron of the zinc finger protein 223 (ZNF223) gene is located (Table 1).…”

Section: Resultsmentioning

confidence: 97%

“…1073/pnas.1216894110/-/DCSupplemental. a second plasmid that expresses ϕC31 integrase (27). Under our assay conditions, replication occurs while the lesion is in the chromosomal locus into which the plasmid had integrated.…”

Section: Resultsmentioning

confidence: 99%

“…In the second step, an attB recognition sequence was added to pSKSL-puro. The attB recognition sequence was obtained from the plasmid pTA-attB (27) by PCR amplification, using the primers 5′-GAGCGG-CCGCCAGTGTGATGGAGATCTGCA-3′ (containing a BstXI site) and 5′-CTAGTAAC GGC CGCCACACCGCTGGAATTC-3′ (containing a BglII site). The PCR product (374 bp) was cut with BstXI and BglII and ligated to BstXI-BglII cut-modified pSKSLpuro.…”

Section: Methodsmentioning

confidence: 99%

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Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells

Izhar

Ziv

Cohen

et al. 2013

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

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DNA lesions can block replication forks and lead to the formation of single-stranded gaps. These replication complications are mitigated by DNA damage tolerance mechanisms, which prevent deleterious outcomes such as cell death, genomic instability, and carcinogenesis. The two main tolerance strategies are translesion DNA synthesis (TLS), in which low-fidelity DNA polymerases bypass the blocking lesion, and homology-dependent repair (HDR; postreplication repair), which is based on the homologous sister chromatid. Here we describe a unique high-resolution method for the simultaneous analysis of TLS and HDR across defined DNA lesions in mammalian genomes. The method is based on insertion of plasmids carrying defined site-specific DNA lesions into mammalian chromosomes, using phage integrase-mediated integration. Using this method we show that mammalian cells use HDR to tolerate DNA damage in their genome. Moreover, analysis of the tolerance of the UV light-induced 6-4 photoproduct, the tobacco smokeinduced benzo[a]pyrene-guanine adduct, and an artificial trimethylene insert shows that each of these three lesions is tolerated by both TLS and HDR. We also determined the specificity of nucleotide insertion opposite these lesions during TLS in human genomes. This unique method will be useful in elucidating the mechanism of DNA damage tolerance in mammalian chromosomes and their connection to pathological processes such as carcinogenesis.error-prone DNA repair | homologous recombination repair | recombinational repair D NA damage is abundant, caused by both external agents such as sunlight and tobacco smoke and intracellular byproducts of metabolism, amounting to about 50,000 lesions per day per cell (1). Despite the presence of effective DNA repair mechanisms that eliminate lesions and restore the original DNA sequence, DNA replication often encounters unrepaired lesions that have escaped repair. These DNA damages may cause arrest of replication forks and the generation of postreplication gaps (2, 3). To complete replication and prevent the formation of double-strand breaks, which are highly deleterious, cells use DNA damage tolerance (DDT) mechanisms. These include translesion DNA synthesis (TLS) and homology-dependent repair (HDR), which enable bypass of the lesions and completion of replication, without removing the lesions from DNA. HDR uses the sequence from the intact sister chromatid to patch the single-stranded template region carrying the lesion. [We term HDR the pathways of DNA damage tolerance that rely on the homologous sister chromatid, also termed postreplication repair (PRR), damage avoidance, template switch, copy choice recombination, and homologous recombination repair.] This is carried out either by physical transfer of the segment complementary to the damaged template [also termed homologous recombination repair (HRR)] or by copying the complementary strand from the sister chromatid (template switch or postreplication repair). TLS employs specialized low-fidelity DNA polymerases to replicate across ...

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells

Izhar

Ziv

Cohen

et al. 2013

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

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“…3B) is similar to that of the C31 integrase (0.7%) (31). C31 is a member of the family of large serine recombinases and has received substantial attention because of the advantage of this enzyme to mediate unidirectional integration (6). It remains to be determined why these 2 reactions, one irreversible ( C31) and the other kinetically disfavored (GinC5), yield similar integrative efficiencies.…”

Section: Discussionmentioning

confidence: 94%

“…Scientists in biology, biotechnology, stem cell research, and gene therapy currently rely on naturally occurring enzymes to perform functions like DNA integration and excision. However, these enzymes recognize multiple sites within the human genome, often resulting in off-target DNA integration and chromosomal translocation (2)(3)(4)(5)(6). Our recent work with serine resolvases and invertases led us to hypothesize that we could use a modular approach that capitalizes on cooperative specificity to design synthetic enzymes that would uniquely recognize a single site within the 3 billion-base-pair human genome and allow us to deliver DNA specifically to this site ( Fig.…”

mentioning

confidence: 99%

Synthesis of programmable integrases

Gordley

Gersbach

Barbas

2009

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

104

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Accurate modification of the 3 billion-base-pair human genome requires tools with exceptional sequence specificity. Here, we describe a general strategy for the design of enzymes that target a single site within the genome. We generated chimeric zinc finger recombinases with cooperative DNA-binding and catalytic specificities that integrate transgenes with >98% accuracy into the human genome. These modular recombinases can be reprogrammed: New combinations of zinc finger domains and serine recombinase catalytic domains generate novel enzymes with distinct substrate sequence specificities. Because of their accuracy and versatility, the recombinases/integrases reported in this work are suitable for a wide variety of applications in biological research, medicine, and biotechnology where accurate delivery of DNA is desired.recombinases ͉ zinc finger ͉ gene delivery ͉ gene targeting ͉ protein engineering T he postgenomic era of medicine will be defined by our ability to achieve biological control through genetic reprogramming. New tools are needed to accurately rewrite the genomic script and specifically alter genes, gene expression, and epigenetic state at any desired loci. To date, no enzyme-natural or synthetic-has been able to accurately modify only a single targeted site within the human genome (1). Scientists in biology, biotechnology, stem cell research, and gene therapy currently rely on naturally occurring enzymes to perform functions like DNA integration and excision. However, these enzymes recognize multiple sites within the human genome, often resulting in off-target DNA integration and chromosomal translocation (2-6). Our recent work with serine resolvases and invertases led us to hypothesize that we could use a modular approach that capitalizes on cooperative specificity to design synthetic enzymes that would uniquely recognize a single site within the 3 billion-base-pair human genome and allow us to deliver DNA specifically to this site (Fig. 1A) (7).In their native contexts, serine resolvases and invertases selectively recombine target sites within the same DNA molecule. This intramolecular specificity is assured by obligate assembly of large protein complexes, wherein accessory factors bound at neighboring sites impose topological and spatial constraints on the recombination reaction (8). Hyperactive mutants of several serine resolvases and invertases have been discovered that efficiently catalyze unrestricted recombination between minimal dimer-binding sites (Table S1) (9, 10). Furthermore, unlike other site-specific recombinases, serine resolvases and invertases are well suited to synthetic reengineering. These enzymes are modular in both structure and function, each comprised of a distinct catalytic domain flexibly tethered to a small helix-turnhelix DNA-binding domain (DBD). However, these DBDs are poorly suited for accurate genomic recombination (11) because the recognition motifs are short (4-6 bp) and degenerate (12, 13).In contrast, zinc finger DNA-binding proteins recognize target sites of v...

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Site‐specificity of serine integrase demonstrated by the attB sequence preference of ɸBT1 integrase

et al. 2018

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Serine integrases mediate site-specific recombination and are extensively applied in genetic engineering and synthetic biology. However, which regions of the attachment sites determine site-specificity and how these regions function in recombination remain elusive. Here, we explored the sequence features of attB attachment sites recognized by ɸBT1 integrase, a representative serine integrase. A 34-bp DNA motif is found that displays distinct base-specific preference for every position. Further investigation of mutations at different positions within the attB sequence shows different recombination efficiencies and binding affinities. We found four conserved regions within the attB motif that coincide with the results of recombination assays, and mutations in the attB sequence that hamper recombination almost all cause reduced binding affinity.

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Integration Specificity of Phage ϕC31 Integrase in the Human Genome

Cited by 228 publications

References 55 publications

Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells

Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells

Synthesis of programmable integrases

Site‐specificity of serine integrase demonstrated by the attB sequence preference of ɸBT1 integrase

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