Recombination In Mammalian Cells Research Articles

Break-induced replication orchestrates resection-dependent template switching.

Break-induced telomere synthesis (BITS) is a RAD51-independent form of break-induced replication that contributes to alternative lengthening of telomeres1,2. This homology-directed repair mechanism utilizes a minimal replisome comprising proliferating cell nuclear antigen (PCNA) and DNA polymerase-δ to execute conservative DNA repair synthesis over many kilobases. How this long-tract homologous recombination repair synthesis responds to complex secondary DNA structures that elicit replication stress remains unclear3-5. Moreover, whether the break-induced replisome orchestrates additional DNA repair events to ensure processivity is also unclear. Here we combine synchronous double-strand break induction with proteomics of isolated chromatin segments(PICh) to capture the telomeric DNA damage response proteome during BITS1,6. This approach revealed a replication stress-dominated response, highlighted by repair synthesis-driven DNA damage tolerance signalling through RAD18-dependent PCNA ubiquitination. Furthermore, the SNM1A nuclease was identified as the major effector of ubiquitinated PCNA-dependent DNA damage tolerance. SNM1A recognizes the ubiquitin-modified break-induced replisome at damaged telomeres, and this directs its nuclease activity to promote resection. These findings show that break-induced replication orchestrates resection-dependent lesion bypass, with SNM1A nuclease activity serving as a critical effector of ubiquitinated PCNA-directed recombination in mammalian cells.

Synthetic biology approach to developing all-in-one baculovirus vector using mammalian introns and miRNA binding sites

BackgroundInsect baculovirus is a promising vector for gene delivery into mammalian cells. We developed a Cre/loxP-based hybrid baculovirus comprising two viruses: one expressing Cre recombinase and the other harboring the transgene flanked by two loxP sites. Co-transduction of mammalian cells with two viruses confers Cre expression, which excises the loxP-flanking cassette off baculovirus genome and catalyzes DNA minicircle formation, thereby prolonging transgene expression. Two separate baculoviruses avoid undesirable recombination and loss of transgene during virus production process, but reduces gene delivery efficiency and complicates applications. MethodsTo tackle this problem, we exploited synthetic biology strategy to control Cre expression and develop an all-in-one baculovirus harboring both cre and loxP-flanking transgene cassette. We controlled cre transcription with a mammalian EF-1α promoter, and regulated cre translation with an intron inserted within the coding region and bantam miRNA binding site at the 3′UTR. Significant findingsThe all-in-one baculovirus vector selectively conferred Cre/loxP-mediated recombination in mammalian cells, but not in E. coli and Sf-9 cells, hence circumventing transgene excision from the virus genome during gene cloning in E. coli and virus amplification in insect cells. The all-in-on vector enabled formation of DNA minicircle in mammalian cells at efficiencies exceeding 80% and implicated its potential for gene delivery. Our design may be expanded to other gene delivery systems that require two separate vectors and is beneficial to gene therapy applications.

ERCC6L2 promotes DNA orientation-specific recombination in mammalian cells.

Programmed DNA recombination in mammalian cells occurs predominantly in a directional manner. While random DNA breaks are typically repaired both by deletion and by inversion at approximately equal proportions, V(D)J and class switch recombination (CSR) of immunoglobulin heavy chain gene overwhelmingly delete intervening sequences to yield productive rearrangement. What factors channel chromatin breaks to deletional CSR in lymphocytes is unknown. Integrating CRISPR knockout and chemical perturbation screening we here identify the Snf2-family helicase-like ERCC6L2 as one such factor. We show that ERCC6L2 promotes double-strand break end-joining and facilitates optimal CSR in mice. At the cellular levels, ERCC6L2 rapidly engages in DNA repair through its C-terminal domains. Mechanistically, ERCC6L2 interacts with other end-joining factors and plays a functionally redundant role with the XLF end-joining factor in V(D)J recombination. Strikingly, ERCC6L2 controls orientation-specific joining of broken ends during CSR, which relies on its helicase activity. Thus, ERCC6L2 facilitates programmed recombination through directional repair of distant breaks.

The dichotomous effects of caffeine on homologous recombination in mammalian cells

This study was initiated to examine the effects of caffeine on the DNA damage response (DDR) and homologous recombination (HR) in mammalian cells. A 5 mM caffeine treatment caused the cell cycle to stall at G2/M and cells eventually underwent apoptosis. Caffeine exposure also induced a strong DDR along with subsequent activation of wildtype p53 protein. An unexpected observation was the caffeine-induced depletion of Rad51 (and Brca2) proteins. Consequently, caffeine-treated cells were expected to be inefficient in HR. However, a dichotomy in the HR response of cells to caffeine treatment was revealed. Caffeine treatment rendered cells significantly better at performing the nascent DNA synthesis that accompanies the early strand invasion steps of HR. Additionally, caffeine treatment increased chromatin accessibility and elevated the efficiency of illegitimate recombination. Conversely, the increase in nascent DNA synthesis did not translate into a higher number of gene targeting events. Thus, prolonged caffeine exposure stalls the cell cycle, induces a p53-mediated apoptotic response and a down-regulation of critical HR proteins, and for reasons discussed, stimulates early steps of HR, but not the formation of complete recombination products.

ERCC1/XPF Is Important for Repair of DNA Double-Strand Breaks Containing Secondary Structures

SummaryThe structure-specific endonuclease ERCC1/XPF plays an important role in nucleotide excision repair and interstrand cross-link repair. In this study, we identified new functions of ERCC1/XPF in DNA double-strand break (DSB) repair. We found that the conserved function of ERCC1/XPF to remove non-homologous sequences at DSBs is a rate-limiting step for homologous recombination in mammalian cells, and more importantly, we uncovered an indispensable role of ERCC1/XPF in repair of DSBs containing DNA secondary structures, including the structure-prone AT-rich DNA sequences derived from common fragile sites and G-quadruplexes (G4s). We also demonstrated a synthetic lethal interaction of XPF with DNA translocase FANCM that is involved in removing DNA secondary structures. Furthermore, inactivation of XPF sensitizes FANCM-deficient cells to G4-interacting compounds. These results suggest an important function of ERCC1/XPF in protecting DNA secondary structures and provide a rationale for targeted treatment of FANCM-deficient tumors through inhibition of XPF.

Mismatch tolerance during homologous recombination in mammalian cells

We investigated the homology dependency of recombination in thymidine kinase (tk)-deficient mouse fibroblasts. Cells were transfected with DNA constructs harboring a herpes tk gene (the “recipient”) rendered non-functional by an oligonucleotide containing the recognition site for endonuclease I-SceI. Constructs also contained a “donor” tk sequence that could restore function to the recipient gene through spontaneous gene conversion or via repair of a double-strand break (DSB) at the I-SceI site. Recombination events were recoverable by selection for tk-positive clones. Three different donors were used containing 16, 25, or 33 mismatches relative to the recipient. The mismatches were clustered, forming an interval of “homeology” relative to the recipient sequences. We show that when homeologous sequences were surrounded by high homology, mismatches were frequently included in gene conversion events. Notably, conversion tracts from spontaneous recombination included either all or none of the mismatches, suggesting that recombination must begin and end in high homology. This requirement was relaxed for events that occurred near an induced DSB, as a significant number of these latter conversion tracts had one end positioned within homeology. Knock-down of mismatch repair showed that incorporation of mismatches into gene conversion tracts can involve repair of mismatched heteroduplex intermediates, indicating that mismatch repair does not necessarily impede homeologous genetic exchange. Our results illustrate (1) genetic exchange between homeologous sequences in a mammalian genome is enabled by nearby homology, (2) proximity to a DSB impacts the homology requirements for where genetic exchange may begin and end, and (3) mismatch correction and previously documented anti-recombination activity are separable functions of the mismatch repair machinery in mammalian cells.

Role of Homology and DNA Double‐Strand Breaks in Initiation of Intrachromosomal Recombination in Mammalian Cells

As part of our continuing investigations into the mechanisms of intrachromosomal recombination in mammalian cells, we stably transfected thymidine kinase deficient mouse fibroblasts with a DNA construct, pKM1, harboring a herpes thymidine kinase (tk) gene rendered nonfunctional by insertion of an oligonucleotide containing the recognition site for endonuclease I‐SceI. We refer to this latter tk sequence as the “recipient.” The construct also contained a closely linked truncated “donor” tk sequence. The recipient and donor were positioned as direct repeats with respect to transcriptional orientation. The donor could potentially restore function to the recipient gene via recombination with the recipient, and such events could be provoked by induction of a double‐strand break (DSB) at the I‐SceI site in the recipient. Such DSB repair events were recoverable by selection for tk‐positive clones. The donor contained 33 mismatches relative to the recipient. The mismatches were clustered, forming a localized segment of DNA sequence displaying about 20% divergence relative to the recipient, and the mismatched segment was surrounded by regions of high homology. When the donor was aligned with the recipient in pKM1, the DSB site in the recipient aligned opposite the mismatched segment, allowing us to potentially capture recombinational repair events initiating between diverged sequences. Our earlier work demonstrated that mammalian cells effectively avoid recombination between 20% diverged sequences. In work presented here, we asked whether flanking regions of high homology would enable genetic exchange between highly diverged sequences or, instead, would rejection of exchange between diverged sequences remain unchanged. We found that by surrounding mismatches with high homology, suppression of recombination between diverged sequences was overcome. Strikingly, we recovered a high frequency of gene conversion tracts positioned entirely within the mismatched sequences. We infer that such events were enabled by homologous pairing interactions between sequences surrounding the site of strand invasion. Our results suggest that the search for high homology prior to recombination is not mediated by an invading DNA terminus and may actually be independent of DSB formation. Further, the homology that enabled the recovered recombination events was located at a discrete distance from the actual invading DNA terminus. In an extension of our studies, we designed two additional constructs, pCW1 and pCW2. These constructs were similar to pKM1 except the recipient sequence in each was disrupted by an 8bp XhoI linker insertion and each construct contained a I‐SceI recognition site positioned within vector sequences at a location outside of either the recipient or donor sequence. Cells transfected with constructs pCW1 and pCW2 are being used to further explore whether a chromosomal DSB can induce and/or influence the nature of genetic exchanges occurring at a discrete distance from the DSB when the genetic exchanges themselves do not directly repair the DSB. Progress addressing this latter issue is presented.Support or Funding InformationThis work was supported by grant MCB 1157416 from the National Science Foundation to ASW and BCW.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oliver Smithies, DPhil

Dr Oliver Smithies, Weatherspoon Eminent Distinguished Professor of Pathology and Laboratory Medicine at the University of North Carolina, Chapel Hill, passed away on January 10 after a short illness at the age of 91. Dr Smithies made many contributions to biomedical science during his more than 60 years at the laboratory bench but is best known for his contribution to developing the concepts and operative techniques that enabled thousands of laboratories to carry out targeted, homologous recombination in mammalian cells, enabling targeted genetic modifications in mice. The ability to rapidly effect directed changes at the genetic site of one’s choice opened a golden age for establishing cause-and-effect relationships for normal and mutated genes. Indeed, the sequences that both control gene activity and modulate the protein complement of different cells were suddenly amenable to mechanistic dissection. The development of gene targeting via homologous recombination led to his sharing the Nobel Prize in Physiology or Medicine in 2007, with Mario Capecchi, Howard Hughes Medical Institute and University of Utah, and Sir Martin Evans, University of Cambridge, United Kingdom. The Nobel was only one of many prestigious honors awarded to Dr Smithies during his long career. He was elected to the United States National Academy of Sciences in 1971 and inducted into the American Academy of Arts and Sciences in 1978. He won the Albert Lasker Award for Basic Medical Research in 2001; again along with Drs Capecchi and Evans, the Alfred P. Sloan, Jr Prize from the General Motors Foundation; jointly with Capecchi in 1994, the March of Dimes Prize in Developmental Biology; again jointly with Capecchi in 2005; and the Thomas Hunt Morgan Medal in 2007. A complete listing of all his awards and honors, regional, national, and international would fill this entire In Memoriam , but suffice …

A novel reverse genetics system for production of infectious West Nile virus using homologous recombination in mammalian cells

Reverse genetics systems facilitate investigation of many aspects of the life cycle and pathogenesis of viruses. However, genetic instability in Escherichia coli has hampered development of a reverse genetics system for West Nile virus (WNV). In this study, we developed a novel reverse genetics system for WNV based on homologous recombination in mammalian cells. Introduction of the DNA fragment coding for the WNV structural protein together with a DNA-based replicon resulted in the release of infectious WNV. The growth rate and plaque size of the recombinant virus were almost identical to those of the parent WNV. Furthermore, chimeric WNV was produced by introducing the DNA fragment coding for the structural protein and replicon plasmid derived from various strains. Here, we report development of a novel system that will facilitate research into WNV infection.

Complex Breakpoints and Template Switching Associated with Non-canonical Termination of Homologous Recombination in Mammalian Cells.

A proportion of homologous recombination (HR) events in mammalian cells resolve by “long tract” gene conversion, reflecting copying of several kilobases from the donor sister chromatid prior to termination. Cells lacking the major hereditary breast/ovarian cancer predisposition genes, BRCA1 or BRCA2, or certain other HR-defective cells, reveal a bias in favor of long tract gene conversion, suggesting that this aberrant HR outcome might be connected with genomic instability. If termination of gene conversion occurs in regions lacking homology with the second end of the break, the normal mechanism of HR termination by annealing (i.e., homologous pairing) is not available and termination must occur by as yet poorly defined non-canonical mechanisms. Here we use a previously described HR reporter to analyze mechanisms of non-canonical termination of long tract gene conversion in mammalian cells. We find that non-canonical HR termination can occur in the absence of the classical non-homologous end joining gene XRCC4. We observe obligatory use of microhomology (MH)-mediated end joining and/or nucleotide addition during rejoining with the second end of the break. Notably, non-canonical HR termination is associated with complex breakpoints. We identify roles for homology-mediated template switching and, potentially, MH-mediated template switching/microhomology-mediated break-induced replication, in the formation of complex breakpoints at sites of non-canonical HR termination. This work identifies non-canonical HR termination as a potential contributor to genomic instability and to the formation of complex breakpoints in cancer.

Phosphorylation of Bovine Herpesvirus 1 VP8 Plays a Role in Viral DNA Encapsidation and Is Essential for Its Cytoplasmic Localization and Optimal Virion Incorporation.

VP8 is a major tegument protein of bovine herpesvirus 1 (BoHV-1) and is essential for viral replication in cattle. The protein undergoes phosphorylation after transcription through cellular casein kinase 2 (CK2) and a viral kinase, US3. In this study, a virus containing a mutated VP8 protein that is not phosphorylated by CK2 and US3 (BoHV-1-YmVP8) was constructed by homologous recombination in mammalian cells. When BoHV-1-YmVP8-infected cells were observed by transmission electron microscopy, blocking phosphorylation of VP8 was found to impair viral DNA encapsidation, resulting in release of incomplete viral particles to the extracellular environment. Consequently, less infectious virus was produced by the mutant virus than by wild-type (WT) virus. A comparison of mutant and WT VP8 by confocal microscopy revealed that mutant VP8 is nuclear throughout infection while WT VP8 is nuclear early during infection and is associated with the Golgi apparatus at later stages. This, together with the observation that mutant VP8 is present in virions, albeit in smaller amounts, suggests that the incorporation of VP8 may occur at two stages. The first takes place without the need for phosphorylation and before or during nuclear egress of capsids, whereas the second occurs in the Golgi apparatus and requires phosphorylation of VP8. The results indicate that phosphorylated VP8 plays a role in viral DNA encapsidation and in the secondary virion incorporation of VP8. To perform these functions, the cellular localization of VP8 is adjusted based on the phosphorylation status. In this study, phosphorylation of VP8 was shown to have a function in BoHV-1 replication. A virus containing a mutated VP8 protein that is not phosphorylated by CK2 and US3 (BoHV-1-YmVP8) produced smaller numbers of infectious virions than wild-type (WT) virus. The maturation and egress of WT and mutant BoHV-1 were studied, showing a process similar to that reported for other alphaherpesviruses. Interestingly, lack of phosphorylation of VP8 by CK2 and US3 resulted in reduced incorporation of viral DNA into capsids during mutant BoHV-1 infection, as well as lower numbers of extracellular virions. Furthermore, mutant VP8 remained nuclear throughout infection, in contrast to WT VP8, which is nuclear at early stages and Golgi apparatus associated late during infection. This correlates with smaller amounts of mutant VP8 in virions and suggests for the first time that VP8 may be assembled into the virions at two stages, with the latter dependent on phosphorylation.

Mismatch Tolerance During Homologous Recombination in Mammalian Cells

We investigated the homology dependency of recombination in cultured thymidine kinase-deficient mouse fibroblasts. Cells were transfected with DNA constructs harboring a herpes tk gene (the “recipient”) which was rendered non-functional by insertion of an oligonucleotide containing the recognition site for endonuclease I-SceI. Constructs also contained a closely linked truncated “donor” tk sequence that could restore function to the recipient gene through spontaneous gene conversion or via recombinational repair provoked by induction of a double-strand break (DSB) at the I-SceI site. Recombination events were recoverable by selection for tk-positive clones. We used three different donor sequences that contained 16, 25, or 33 mismatches relative to the recipient. The mismatches within these “homeologous” sequences were clustered and were surrounded by regions of high homology. Previous work indicated that mammalian cells fastidiously avoid recombination between homeologous sequences. We show that when homeologous sequences are surrounded by high homology, mismatches are frequently included in gene conversion events. Gene conversion tracts from spontaneous recombination revealed that in each event either all or none of the mismatches were transferred from donor to recipient, suggesting that recombination must begin and end in high homology. This requirement was somewhat relaxed for DSB-induced events. Knock-down of mismatch repair provided evidence that incorporation of mismatches into gene conversion tracts involved repair of mismatched heteroduplex intermediates. Our results indicate that mismatch repair of multiple mispaired bases does not function to impede exchange between homeologous sequences. Supported by grant MCB: 1157416 from the NSF to ASW and BCW.

Infectious delivery of alphaherpesvirus bacterial artificial chromosomes.

Bacterial artificial chromosomes (BACs) can accommodate and stably propagate the genomes of large DNA viruses in E. coli. As DNA virus genomes are often per se infectious upon transfection into mammalian cells, their cloning in BACs and easy modification by homologous recombination in bacteria has become an important strategy to investigate the functions of individual virus genes. This chapter describes a strategy to clone the genomes of viruses of the Alphaherpesvirinae subfamily within the family of the Herpesviridae, which is a group of large DNA viruses that can establish both lytic and latent infections in most animal species including humans. The cloning strategy includes the following steps: (1) Construction of a transfer plasmid that contains the BAC backbone with selection and screening markers, and targeting sequences which support homologous recombination between the transfer plasmid and the alphaherpesvirus genome. (2) Introduction of the transfer plasmid sequences into the alphaherpesvirus genome via homologous recombination in mammalian cells. (3) Isolation of recombinant virus genomes containing the BAC backbone sequences from infected mammalian cells and electroporation into E. coli. (4) Preparation of infectious BAC DNA from bacterial cultures and transfection into mammalian cells. (5) Isolation and characterization of progeny virus.

Assaying break and nick-induced homologous recombination in mammalian cells using the DR-GFP reporter and Cas9 nucleases.

Thousands of DNA breaks occur daily in mammalian cells, including potentially tumorigenic double-strand breaks (DSBs) and less dangerous but vastly more abundant single-strand breaks (SSBs). The majority of SSBs are quickly repaired, but some can be converted to DSBs, posing a threat to the integrity of the genome. Although SSBs are usually repaired by dedicated pathways, they can also trigger homologous recombination (HR), an error-free pathway generally associated with DSB repair. While HR-mediated DSB repair has been extensively studied, the mechanisms of HR-mediated SSB repair are less clear. This chapter describes a protocol to investigate SSB-induced HR in mammalian cells employing the DR-GFP reporter, which has been widely used in DSB repair studies, together with an adapted bacterial CRISPR/Cas system.

Impact Of Rad50 On Telomere Recombination Mechanisms In Primary Bone Marrow and B-Cell Lymphomas Lacking Telomerase

The unlimited growth that occurs in tumors requires telomere maintenance. Yet, a portion of human tumors lack telomerase, and maintain telomeres using recombination-based mechanisms. Studies in other model organisms indicate that two different pathways of recombination-based mechanisms impact telomere maintenance and rely on the DNA repair proteins Rad50 and Rad51. In the Rad50-dependent pathway telomere recombination occurs within the telomere repeats. In contrast, recombination using the Rad51-dependent pathway occurs within repetitive sequences in the subtelomeres. Using a mouse B-cell lymphoma model lacking telomerase, Eμmyc+mTR-/-, and immortalized fibroblast cells lacking the RNA component of telomerase (mTR-/-) we have examined the impact of inhibiting Rad50 and Rad51a on telomere recombination. We find inhibiting Rad50 or Rad51a in Eμmyc+mTR-/- B-cell lymphomas, and in mTR-/- immortalized fibroblasts, has a synergistic effect on DNA damage sensitivity to mitomycin but not camptothecin. Inhibiting Rad50 in telomerase deficient cells also results in telomere shortening and in some tumors, reduced growth. In contrast, when Rad50 or Rad51a is inhibited in cells with telomerase, DNA damage sensitivity from mitomycin is not observed when compared to cells expressing a control shRNA. In addition inhibiting Rad50 in cells with telomerase does not significantly impact telomere length or recombination. Next we developed a comparative genomic hybridization (aCGH) approach that detects recombination events in the subtelomeres. Using these subtelomere arrays we find B-cell lymphomas lacking telomerase exhibit a significant increase in subtelomere recombination compared to primary cells. We also examined the impact of inhibiting Rad50 on subtelomere recombination events. Our findings using aCGH suggest that inhibiting Rad50 does not impact subtelomere recombination in Eμmyc+mTR-/- B-cell lymphomas. Overall, our findings suggest that inhibiting either Rad50 or Rad51a in mTR-/- cells has a synergistic impact on the sensitivity to DNA damaging agents in contrast to cells with mTR+/+. Currently we are testing the impact of inhibiting Rad51a on subtelomere recombination. In addition these results further support that Rad50 contributes to telomere recombination mechanisms in tumors lacking telomerase and will provide insight into the mechanism of subtelomere recombination in mammalian cells. Disclosures:No relevant conflicts of interest to declare.

FBH1 Helicase Disrupts RAD51 Filaments in Vitro and Modulates Homologous Recombination in Mammalian Cells

Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.

Efficient reversal of phiC31 integrase recombination in mammalian cells

Over the past decade, the integrase enzyme from phage phiC31 has proven to be a useful genome engineering tool in a wide variety of species, including mammalian cells. The enzyme efficiently mediates recombination between two distinct sequences, attP and attB, producing recombinant product sites, attL and attR. The reaction proceeds exclusively in a unidirectional manner, because integrase is unable to synapse attL and attR. To date, use of phiC31 integrase has been limited to attP × attB recombination. The factor needed for the reverse reaction--the excisionase or recombination directionality factor (RDF)--was identified recently and shown to function in vitro and in bacterial cells. To determine whether the phiC31 RDF could also function in mammalian cells, we cloned and tested several vectors that permit assessment of phiC31 RDF activity in mammalian environments. In the human and mouse cell lines tested (HeLa, HEK293, and NIH3T3), we observed robust RDF activity, using plasmid and/or genomic assays. This work is the first to demonstrate attL-attR serine integrase activity in mammalian cells and validates phiC31 RDF as a new tool that will enable future studies to take advantage of phiC31 integrase recombination in the forward or reverse direction.

A Method for Producing Transgenic Cells Using a Multi-Integrase System on a Human Artificial Chromosome Vector

The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3–96.8%. Additionally, we observed homogenous gene expression in 77.3–87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.

The XPD subunit of TFIIH is required for transcription-associated but not DNA double-strand break-induced recombination in mammalian cells

Mutations in the XPD gene can give rise to three phenotypically distinct disorders: xeroderma pigmentosum (XP), trichothiodystrophy (TTD) or combined XP and Cockayne syndrome (CS) (XP/CS). The role of Xeroderma Pigmentosum group D protein (XPD) in nucleotide excision repair explains the increased risk of skin cancer in XP patients but not all the clinical phenotypes found in XP/CS or TTD patients. Here, we describe that the XPD-defective UV5 cell line is impaired in transcription-associated recombination (TAR), which can be reverted by the introduction of the wild-type XPD gene expressed from a vector. UV5 cells are defective in TAR, despite having intact transcription and homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Interestingly, we find reduced spontaneous HR in XPD-defective cells, suggesting that transcription underlies a portion of spontaneous HR events. We also report that transcription-coupled repair (TCR)-defective cells, mutated in the Cockayne syndrome B (CSB) protein, have a defect in TAR, but not in DSB-induced HR. However, the TAR defect may be associated with a general transcription defect in CSB-deficient cells. In conclusion, we show a novel role for the XPD protein in TAR, linking TAR with TCR.

Critical Amino Acid Residues Within the ɸC31 Integrase DNA-Binding Domain Affect Recombination Activities in Mammalian Cells

The bacteriophage-derived ϕC31 integrase system represents an attractive tool for site-directed recombination in mammalian cells. Its integration reaction is based on recombination between the attachment site attB within an episomal substrate plasmid and either the bacteriophage-derived wild-type attachment site attP or pseudo-attP attachment sites (attP') present in the mammalian genome. In the present study we aimed at increasing the safety and efficiency of ϕC31 integrase-mediated recombination by mutating the DNA-binding domain located at the C terminus. Using an alanine mutagenesis approach, we generated 22 ϕC31 point mutants that were screened for activities in mammalian cells. Intramolecular excision assays based on recombination between attB and attP revealed five mutants with 2-fold enhanced excision activity. Importantly, we also identified mutants showing enhanced recombination activities between attB and three previously described attP' sites detected in the mammalian genome, indicating that there may be enhanced specificity for these hot spots. Several mutants showed, in mammalian cells, integration activities that increased in a cell line-dependent manner. The combination of beneficial mutations in addition to optimization of the integrase plasmid dose enhanced integration efficiencies up to 5.5-fold. We also identified three ϕC31 integrase mutants that were recombination defective in all applied assays, suggesting that these amino acid residues are essential for the functionality of ϕC31 integrase in mammalian cells. In summary, we identified critical amino acid residues within the ϕC31 DNA-binding domain. With respect to site-directed recombination and genome engineering these findings have important implications for improved ϕC31 protein design.