Efficient integration of short interspersed element-flanked foreign DNA via homologous recombination

We investigated whether mouse short interspersed elements (SINEs) could influence the recombination frequency of foreign DNA. Vectors harboring a reporter gene in combinations of SINEs B1 and/or B2 or a portion of long interspersed element-1 were prepared and tested in vitro by a colony assay using HC11 murine mammary epithelial cells and in vivo by microinjection into fertilized mouse eggs. In transfected HC11 cells, the number of colonies surviving G418 selection increased by 3.5-fold compared with control when the reporter was flanked by fused B1-B2 sequences. Similar results were obtained from microinjection study; in fetuses 11.5 days post coitum, transgene positives in control and SINE-flanked vectors were 16 and 53%, respectively. Individual B1- and B2-harboring vectors showed equivalent activities with each other, as determined by the colony assay (2.8-fold versus 3.2-fold compared with control). We determined the contribution of homologous recombination to the SINE-mediated increase in integration frequency through a polymerase chain reaction-based strategy; in more than half of embryos transgenes underwent homologous recombinations involving B1 sequences. These results demonstrate that the SINE sequences can increase the integration rate of foreign DNA and that such an increase is most likely due to the enhancement of homologous recombination.     

Increase in Ty1 cDNA recombination in yeast sir4 mutant strains at high temperature

Transposition of the Ty1 element of the yeast Saccharomyces cerevisiae is temperature sensitive. We have identified a null allele of the silent information regulator gene SIR4 as a host mutant that allows for transposition at high temperature. We show that the apparent increase in transposition activity in sir4 mutant strains at high temperature is dependent on the RAD52 gene and is thus likely resulting from an increase in Ty1 cDNA recombination, rather than in IN-mediated integration. General cellular recombination is not increased at high temperature, suggesting that the increase in recombination at high temperature in sir4 mutants is specific for Ty1 cDNA. Additionally, this high-temperature Ty1 recombination was found to be dependent on functional Sir2p and Sir3p. We speculate that the increase in recombination seen in sir4 mutants at high temperature may be due to changes in chromatin structure or Ty1 interactions with chromosomal structures resulting in higher recombination rates.     

Efficient nonhomologous and homologous recombination in scid cells

The severe combined immunodeficiency (scid) mutation affects both coding joint formation during immunoglobulin and T-cell receptor V(D)J recombination and double-strand break repair. We analyzed scid cells for their ability to undergo other types of DNA end joining: non-homologous and homologous recombination. Using plasmid constructs carrying antibiotic resistance genes, we observed that the efficiency of nonhomologous integration in scid cells was equal to that in wildtype cell lines. In addition, there was no obvious difference in the fidelity of the integration and in the expression of the resistance genes. Moreover, scid cells were able to carry out homologous recombination of extrachromosomal substrates just as well as wildtype cells. These results suggest a mechanistic difference between nonhomologous integration and homologous recombination on the one hand and V(D)J recombination and double-strand break repair on the other.     

Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants

Chromatin assembly factor 1 (CAF-1) is involved in nucleo some assembly following DNA replication and nucleotide excision repair. In Arabidopsis thaliana, the three CAF-1 subunits are encoded by FAS1, FAS2 and, most likely, MSI1, respectively. In this study, we asked whether genomic stability is altered in fas1 and fas2 mutants that are lacking CAF-1 activity. Depletion of either subunit increased the frequency of somatic homologous recombination (HR) in planta approximately 40-fold. The frequency of transferred DNA (T-DNA) integration was also elevated. A delay in loading histones onto newly replicated or repaired DNA might make these DNA stretches more accessible, both to repair enzymes and to foreign DNA. Furthermore, fas mutants exhibited increased levels of DNA double-strand breaks, a G2-phase retardation that accelerates endoreduplication, and elevated levels of mRNAs coding for proteins involved in HR-all factors that could also contribute to upregulation of HR frequency in fas mutants.     

Efficient gene targeting by homologous recombination in rice

Modification of genes through homologous recombination, termed gene targeting, is the most direct method to characterize gene function. In higher plants, however, the method is far from a common practice. Here we describe an efficient and reproducible procedure with a strong positive/negative selection for gene targeting in rice, which feeds more than half of the world's population and is an important model plant. About 1% of selected calli and their regenerated fertile plants were heterozygous at the targeted locus, and only one copy of the selective marker used was found at the targeted site in their genomes. The procedure's applicability to other genes will make it feasible to obtain various gene-targeted lines of rice.     

Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination

An efficient method is described for the generation of site-specific chromosomal integrations in Lactobacillus acidophilus and Lactobacillus gasseri. The strategy is an adaptation of the lactococcal pORI system (K. Leenhouts, G. Venema, and J. Kok, Methods Cell Sci. 20:35-50, 1998) and relies on the simultaneous use of two plasmids. The functionality of the integration strategy was demonstated by the insertional inactivation of the Lactobacillus acidophilus NCFM lacL gene encoding beta-galactosidase and of the Lactobacillus gasseri ADH gusA gene encoding beta-glucuronidase.     

Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli.

Despite recent technical improvements, the construction of recombinant adenovirus vectors remains a time-consuming procedure which requires extensive manipulations of the viral genome in both Escherichia coli and eukaryotic cells. This report describes a novel system based on the cloning and manipulation of the full-length adenovirus genome as a stable plasmid in E. coli, by using the bacterial homologous recombination machinery. The efficiency and flexibility of the method are illustrated by the cloning of the wild-type adenovirus type 5 genome, the insertion of a constitutive promoter upstream from the E3 region, the replacement of the E1 region by an exogenous expression cassette, and the deletion of the E1 region. All recombinant viral DNAS were shown to be fully infectious in permissive cells, and the modified E3 region or the inserted foreign gene was correctly expressed in the infected cells.     

Kluyveromyces lactis rDNA as a target for multiple integration by homologous recombination.

Gene targeting to a single chromosomal locus has been extensively used in Saccharomyces cerevisiae. In this study, we have analyzed targeting of a repetitive sequence, the 25S rDNA gene, to the chromosomal rDNA cluster of Kluyveromyces lactis by the use of a replacement vector. We have obtained K. lactis transformants carrying multiple copies of the replacement cassette inserted into the rDNA chromosomal locus. Analysis of several transformants has shown that the number of integrated copies could range from 4 to 40. Moreover, the distribution of integration sites within the rDNA locus was found to differ in most transformants. Single-copy integration at multiple sites, rather than multicopy integration at a very limited number of sites, was found to be the most frequent event. Also, in most transformants, integration sites were distributed at random as well as in an orderly fashion, i.e., in contiguous or alternate rDNA repeats, suggesting that amplification of the integrated sequences, rather than multiple integration events, may account for the copy number of insertions.     

Sws1 is a conserved regulator of homologous recombination in eukaryotic cells

Rad52-dependent homologous recombination (HR) is regulated by the antirecombinase activities of Srs2 and Rqh1/Sgs1 DNA helicases in fission yeast and budding yeast. Functional analysis of Srs2 in Schizosaccharomyces pombe led us to the discovery of Sws1, a novel HR protein with a SWIM-type Zn finger. Inactivation of Sws1 suppresses the genotoxic sensitivity of srs2Delta and rqh1Delta mutants and rescues the inviability of srs2Delta rqh1Delta cells. Sws1 functions at an early step of recombination in a pro-recombinogenic complex with Rlp1 and Rdl1, two RecA-like proteins that are most closely related to the human Rad51 paralogs XRCC2 and RAD51D, respectively. This finding indicates that the XRCC2-RAD51D complex is conserved in lower eukaryotes. A SWS1 homolog exists in human cells. It associates with RAD51D and ablating its expression reduces the number of RAD51 foci. These studies unveil a conserved pathway for the initiation and control of HR in eukaryotic cells.     

A Ty1 cell-type-specific regulatory sequence is a recognition element for a constitutive binding factor.

Ty transposable-element insertion mutations of Saccharomyces cerevisiae can cause cell-type-dependent activation of adjacent-gene expression. Several cis-acting regulatory regions within Ty1 are responsible for the effect of Ty1 on adjacent-gene expression. One of these is the block II sequence that was defined by its homology to mammalian enhancers and to the yeast a1-alpha 2 control site. Tandem copies of a 57-base-pair region encompassing block II caused an additive increase in expression of the CYC7 reporter gene in the absence of other Ty1 sequences. The activation of gene expression by the multiple repeats was abolished in a/alpha diploid cells. A specific complex between a constitutive factor in whole-cell extracts and the DNA regulatory element was observed. The protein-binding site for the constitutive factor coincided with the block II element. Base-pair substitutions within the binding site abolished the ability of the block II element to function as a component of the Ty1 activator and to form the factor-DNA complex. The correlation between complex formation and reporter gene expression indicates that factor binding to the cis-acting element is essential for this element to function as a component of the Ty1 activator.     

Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination

Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR–Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR–CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.     

Correct integration of model substrates by Ty1 integrase

The retrovirus-like mobile genetic element of Saccharomyces cerevisiae, Ty1, transposes to new genomic locations via the element-encoded integrase (IN). Here we report that purified recombinant IN catalyzed correct integration of a linear DNA into a supercoiled target plasmid. Ty1 virus-like particles (VLPs) integrated donor DNA more efficiently than IN. VLP and IN-mediated insertions occurred at random sites in the target. Mg(2+) was preferred over Mn(2+) for correct integration, and neither cation enhanced nonspecific nuclease activity of IN. Products consistent with correct integration events were also obtained by Southern analysis. Recombinant IN and VLPs utilized many, but not all, linear donor fragments containing non-Ty1 ends, including a U3 mutation which has been shown to be defective for transposition in vivo. Together, our results suggest that IN is sufficient for Ty1 integration in vitro and IN interacts with exogenous donors less stringently than with endogenous elements.     

Efficient homologous recombination of linear DNA substrates after injection into Xenopus laevis oocytes.

When DNA molecules are injected into Xenopus oocyte nuclei, they can recombine with each other. With bacteriophage lambda DNAs, it was shown that this recombination is stimulated greatly by introduction of double-strand breaks into the substrates and is dependent on homologous overlaps in the recombination interval. With plasmid DNAs it was shown that little or no recombination occurs between circular molecules but both intra- and intermolecular events take place very efficiently with linear molecules. As with the lambda substrates, homology was required to support recombination; no simple joining of ends was observed. Blockage of DNA ends with nonhomologous sequences interfered with recombination, indicating that ends are used directly to initiate homologous interactions. These observations are combined to evaluate possible models of recombination in the oocytes. Because each oocyte is capable of recombining nanogram quantities of linear DNA, this system offers exceptional opportunities for detailed molecular analysis of the recombination process in a higher organism.     

Hdf1, a yeast Ku-protein homologue, is involved in illegitimate recombination, but not in homologous recombination.

Hdf1 is the yeast homologue of the mammalian 70 kDa subunit of Ku-protein, which has DNA end-binding activity and is involved in DNA double-strand break repair and V(D)J recombination. To examine whether Hdf1 is involved in illegitimate recombination, we have measured the rate of deletion mutation caused by illegitimate recombination on a plasmid in an hdf1 disruptant. The hdf1 mutation reduced the rate of deletion formation by 20-fold, while it did not affect mitotic and meiotic homologous recombinations between two heteroalleles or homologous recombination between direct repeats. Hence Hdf1 participates in illegitimate recombination, but not in homologous recombination, in contrast to Rad52, Rad50, Mre11 and Xrs2, which are involved in both homologous and illegitimate recombination. The illegitimate recombination in the hdf1 disruptant took place between recombination sites that shared short regions of homology (1-4 bp), as was observed in the wild-type. Based on the DNA end-binding activity of Hdf1, we discuss models in which Hdf1 plays an important role in the late step of illegitimate recombination.     

Role of SIRT1 in homologous recombination

The class III histone deacetylase (HDAC) SIRT1 plays a role in the metabolism, aging, and carcinogenesis of organisms and regulates senescence and apoptosis in cells. Recent reports revealed that SIRT1 also deacetylates several DNA double-strand break (DSB) repair proteins. However, its exact functions in DNA repair remained elusive. Using nuclear foci analysis and fluorescence-based, chromosomal DSB repair reporter, we find that SIRT1 activity promotes homologous recombination (HR) in human cells. Importantly, this effect is unrelated to functions of poly(ADP-ribose) polymerase 1 (PARP1), another NAD(+)-catabolic protein, and does not correlate with cell cycle changes or apoptosis. Interestingly, we demonstrate that inactivation of Rad51 does not eliminate the effect of SIRT1 on HR. By epistasis-like analysis through knockdown and use of mutant cells of distinct SIRT1 target proteins, we show that the non-homologous end joining (NHEJ) factor Ku70 as well as the Nijmegen Breakage Syndrome protein (nibrin) are not needed for this SIRT1-mediated effect, even though a partial contribution of nibrin cannot be excluded. Strikingly however, the Werner helicase (WRN), which in its mutated form causes premature aging and cancer and which was linked to the Rad51-independent single-strand annealing (SSA) DSB repair pathway, is required for SIRT1-mediated HR. These results provide first evidence that links SIRT1's functions to HR with possible implications for genomic stability during aging and tumorigenesis.     

Efficient in vitro system of homologous recombination in brome mosaic bromovirus

Recent in vivo studies have revealed that the subgenomic promoter (sgp) in brome mosaic bromovirus (BMV) RNA3 supports frequent homologous recombination events (R. Wierzchoslawski, A. Dzianott, and J. Bujarski, J. Virol. 78:8552-8564, 2004). In this paper, we describe an sgp-driven in vitro system that supports efficient RNA3 crossovers. A 1:1 mixture of two (-)-sense RNA3 templates was copied with either a BMV replicase (RdRp) preparation or recombinant BMV protein 2a. The BMV replicase enzyme supported a lower recombination frequency than 2a, demonstrating a role of other viral and/or host factors. The described in vitro system will allow us to study the mechanism of homologous RNA recombination.     

Ectopic recombination between Ty elements in Saccharomyces cerevisiae is not induced by DNA damage.

Mitotic recombination is increased when cells are treated with a variety of physical and chemical agents that cause damage to their DNA. We show here, using Saccharomyces cerevisiae strains that carry marked Ty elements, that recombination between members of this family of retrotransposons is not increased by UV irradiation or by treatment with the radiomimetic drug methyl methanesulfonate. Both ectopic recombination and mutation events were elevated by these agents for non-Ty sequences in the same strain. We discuss possible mechanisms that can prevent the induction of recombination between Ty elements.