The effect of the Saccharomyces cerevisiae endo-exonuclease NUD1 gene expression on the resistance of HeLa cells to DNA-damaging agents.

HeLa cells transiently transfected with a mammalian expression DNA vector expressing the Saccharomyces cerevisiae endo-exonuclease (EE) NUD1 gene have exhibited changes in cell survival frequencies after treatment with different DNA-damaging agents as compared to HeLa cells transfected with a control plasmid. The NUD1-transfected cells showed a dose-dependent increase in sensitivity to UV irradiation resulting in up to 58% decrease in cell survival. In response to gamma-irradiation NUD1 transfected cells featured an increased survival at doses equal to and greater than 2.0 Gy, reaching a maximum enhancement in survival frequency of 17%. At the same time, the NUD1-transfectants featured an increase in resistance to 0.25 microM-0.5 microM cis-platin (up to 58% increase in cell survival) and 1.0 mM EMS (11% increase). At higher concentrations of EMS NUD1 expression resulted in a decreased cell survival of the transfected cells (17% decrease for 2.5 mM EMS). No difference in cell survival frequencies between the NUD1-transfectants and the controls was observed after treatment with different concentrations of chlorambucil and mechlorethamine. These results suggest possible roles played by EEs in different DNA repair pathways--being stimulatory for the repair of certain types of DNA lesions, such as double strand breaks (DSBs), and interfering with the endogenous DNA repair systems for the repair of other types of lesions. Furthermore, these results also provide additional indirect evidence for the role of EEs in homologous recombination.     

Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination.

In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.     

XRCC3 is required for efficient repair of chromosome breaks by homologous recombination

XRCC3 was originally identified as a human gene able to complement the DNA damage sensitivity, chromosomal instability and impaired growth of the mutant hamster cell line irs1SF. More recently, it has been cloned, sequenced and found to bear sequence homology to the highly conserved eukaryotic repair and recombination gene RAD51. The phenotype of irs1SF and the identification of XRCC3 as a member of the RAD51 gene family have suggested a role for XRCC3 in repair of DNA damage by homologous recombination. Homologous recombinational repair (HRR) of a specifically induced chromosomal double-strand break (DSB) was assayed in irs1SF cells with and without transient complementation by human XRCC3. Complementation with XRCC3 increased the frequencies of repair by 34- to 260-fold. The results confirm a role for XRCC3 in HRR of DNA DSB, and the importance of this repair pathway for the maintenance of chromosomal integrity in mammalian cells.     

BRCA1 interacts with dominant negative SWI/SNF enzymes without affecting homologous recombination or radiation-induced gene activation of p21 or Mdm2

BRCA1 is a tumor suppressor gene linked to familial breast and ovarian cancer. The BRCA1 protein has been implicated in a diverse set of cellular functions, including activation of gene expression by the p53 tumor suppressor and control of homologous recombination (HR) during DNA repair. Prior reports have demonstrated that BRCA1 can exist in cells in a complex with the BRG1-based SWI/SNF ATP-dependent chromatin remodeling enzymes and that SWI/SNF components contribute to p53-mediated gene activation. To investigate the link between SWI/SNF function and BRCA1 mediated effects on p53-mediated gene activation and on mechanisms of homologous recombination, we have utilized mammalian cells that inducibly express an ATPase-deficient, dominant negative SWI/SNF enzymes. Mutant SWI/SNF ATPases retain the ability to interact with BRCA1 in cells. We report that expression of dominant negative SWI/SNF enzymes does not affect p53-mediated induction of the p21 cyclin dependent kinase inhibitor or the Mdm2 E3 ubiquitin ligase that regulates p53 in cells exposed to UV or gamma irradiation. Similarly, integration of a reporter that monitors homologous recombination by gene conversion into these cells demonstrated no change in the recombination rate in the absence of functional SWI/SNF enzyme. We conclude that the SWI/SNF chromatin remodeling enzymes may contribute to but are not required for these processes.     

Gene repair in mammalian cells is stimulated by the elongation of S phase and transient stalling of replication forks

The repair of point mutations directed by modified single-stranded DNA oligonucleotides is dependent on the activity of proteins involved in homologous recombination (HR). As a consequence, factors that stimulate homologous recombination, such as double strand breaks, can impact the frequency with which repair occurs. Here, we report that the stalling of replication forks can also activate the gene repair pathway and lead to an enhanced level of nucleotide exchange. The mammalian cell line, DLD-1, containing an integrated mutant eGFP gene, was used as an assay system to explore how replication fork activity affects the overall repair reaction. The addition of 2',3'-dideoxycytidine (ddC), a nucleoside analog that retards the rate of elongation and effectively stalls the replication fork, results in a lengthened S phase and an increased number of gene repair events. This stimulation was reversed when caffeine was added to the reaction at concentrations that block the homologous recombination pathway. In contrast, the nucleoside analog, 1-beta-D-arabinofuranosylcytosine which stops replication in these cells, failed to stimulate the gene repair reaction to any appreciable degree until the block is released and active replication resumes. Furthermore, overexpression of wild-type p53 which is known to bind transiently to stalled replication forks blocked the stimulatory effect of ddC. Overexpression of mutant p53 genes, deficient in the capacity to bind DNA, however, did not inhibit the reaction. Our results indicate that an expansion of S phase and a transient stalling of replication forks can increase the frequency of targeted gene repair.     

Repair of double-strand breaks by homologous recombination in mismatch repair-defective mammalian cells

Chromosomal double-strand breaks (DSBs) stimulate homologous recombination by several orders of magnitude in mammalian cells, including murine embryonic stem (ES) cells, but the efficiency of recombination decreases as the heterology between the repair substrates increases (B. Elliott, C. Richardson, J. Winderbaum, J. A. Nickoloff, and M. Jasin, Mol. Cell. Biol. 18:93-101, 1998). We have now examined homologous recombination in mismatch repair (MMR)-defective ES cells to investigate both the frequency of recombination and the outcome of events. Using cells with a targeted mutation in the msh2 gene, we found that the barrier to recombination between diverged substrates is relaxed for both gene targeting and intrachromosomal recombination. Thus, substrates with 1.5% divergence are 10-fold more likely to undergo DSB-promoted recombination in Msh2(-/-) cells than in wild-type cells. Although mutant cells can repair DSBs efficiently, examination of gene conversion tracts in recombinants demonstrates that they cannot efficiently correct mismatched heteroduplex DNA (hDNA) that is formed adjacent to the DSB. As a result, >20-fold more of the recombinants derived from mutant cells have uncorrected tracts compared with recombinants from wild-type cells. The results indicate that gene conversion repair of DSBs in mammalian cells frequently involves mismatch correction of hDNA rather than double-strand gap formation. In cells with MMR defects, therefore, aberrant recombinational repair may be an additional mechanism that contributes to genomic instability and possibly tumorigenesis.     

Essential roles of the chromatin remodeling factor BRG1 in spermatogenesis in mice

Mammalian spermatogenesis is a complex process that involves spatiotemporal regulation of gene expression and meiotic recombination, both of which require the modulation of chromatin structure. Proteins important for chromatin regulation during spermatogenesis remain poorly understood. Here we addressed the role of BRG1, the catalytic subunit of the mammalian Swi/Snf-like BAF chromatin-remodeling complex, during spermatogenesis in mice. BRG1 expression is dynamically regulated in the male germline, being weakly detectable in spermatogonia, highly expressed in pachytene spermatocytes, and turned off in maturing round spermatids. This expression pattern overlaps that of Brm, the Brg1 homolog. While Brm knockout males are known to be fertile, germline-specific Brg1 deletion completely arrests spermatogenesis at the midpachytene stage, which is associated with spermatocyte apoptosis and apparently also with impaired homologous recombination and meiotic sex chromosome inactivation. However, Brg1 is dispensable for gammaH2AX formation during meiotic recombination, contrary to its reported role in DNA repair in somatic cells. Our study reveals the essential role of Brg1 in meiosis and underscores the differences in the mechanisms of DNA repair between germ cells and somatic cells.     

Efficient bicistronic expression of cre in mammalian cells

Cre recombinase-mediated DNA recombination is proving to be a powerful technique for the generation of mosaic mutant mice. To develop this technology further, we have altered the cre gene to enhance its expression in mammalian cells and have tested its efficiency of expression in a bicistronic message. Using a transient transfection assay, we found that the extension of a eukaryotic translation initiation consensus sequence, the insertion of two N-terminal amino acids, and the mutation of a cryptic splice acceptor site did not detectably alter Cre recombinase activity. The addition of either of two introns resulted in an approximately 2-fold increase in recombination frequency. We then tested the relative efficacy of Cre-mediated recombination in several bicistronic messages having the encephalomyocarditis virus internal ribosome entry site (IRES). Recombination frequencies were only reduced 2-fold relative to a comparable monocistronic cre gene. The latter results indicate that it will be possible to generate transgenic mouse strains having tissue-specific expression of the Cre recombinase through integration of an IRES-cre gene without disabling the targeted gene.     

Enhancement of Extra Chromosomal Recombination in Somatic Cells by Affecting the Ratio of Homologous Recombination (HR) to Non-Homologous End Joining (NHEJ)

Advancements in somatic cell gene targeting have been slow due to the finite lifespan of somatic cells and the overall inefficiency of homologous recombination. The rate of homologous recombination is determined by mechanisms of DNA repair, and by the balance between homologous recombination (HR) and non-homologous end joining (NHEJ). A plasmid-to-plasmid, extra chromosomal recombination system was used to study the effects of the manipulation of molecules involved in NHEJ (Mre11, Ku70/80, and p53) on HR/NHEJ ratios. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. While a mutant Mre11 and an anti-Ku aptamer did not significantly affect the rate of NHEJ or HR, transient expression of a p53 mutant increased overall HR/NHEJ by 2.5 fold. However, expression of the mutant p53 resulted in increased aneuploidy of the cultured cells. Additionally, we found no relationship between telomerase expression and changes in HR/NHEJ. In contrast, cell synchrony by thymidine incorporation did not induce chromosomal abnormalities, and increased the ratio of HR/NHEJ 5-fold by reducing the overall rate of NHEJ. Overall our results show that attempts at reducing NHEJ by use of Mre11 or anti-Ku aptamers were unsuccessful. Cell synchrony via thymidine incorporation, however, does increase the ratio of HR/NHEJ and this indicates that this approach may be of use to facilitate targeting in somatic cells by reducing the numbers of colonies that need to be analyzed before a HR is identified.     

Enhancement of extra chromosomal recombination in somatic cells by affecting the ratio of homologous recombination (HR) to non-homologous end joining (NHEJ)

Advancements in somatic cell gene targeting have been slow due to the finite lifespan of somatic cells and the overall inefficiency of homologous recombination. The rate of homologous recombination is determined by mechanisms of DNA repair, and by the balance between homologous recombination (HR) and non-homologous end joining (NHEJ). A plasmid-to-plasmid, extra chromosomal recombination system was used to study the effects of the manipulation of molecules involved in NHEJ (Mre11, Ku70/80, and p53) on HR/NHEJ ratios. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. While a mutant Mre11 and an anti-Ku aptamer did not significantly affect the rate of NHEJ or HR, transient expression of a p53 mutant increased overall HR/NHEJ by 2.5 fold. However, expression of the mutant p53 resulted in increased aneuploidy of the cultured cells. Additionally, we found no relationship between telomerase expression and changes in HR/NHEJ. In contrast, cell synchrony by thymidine incorporation did not induce chromosomal abnormalities, and increased the ratio of HR/NHEJ 5-fold by reducing the overall rate of NHEJ. Overall our results show that attempts at reducing NHEJ by use of Mre11 or anti-Ku aptamers were unsuccessful. Cell synchrony via thymidine incorporation, however, does increase the ratio of HR/NHEJ and this indicates that this approach may be of use to facilitate targeting in somatic cells by reducing the numbers of colonies that need to be analyzed before a HR is identified.     

The effects of AtRad52 over‐expression on homologous recombination in Arabidopsis

AtRad52 homologs are involved in DNA recombination and repair, but their precise functions in different homologous recombination (HR) pathways or in gene‐targeting have not been analyzed. In order to facilitate our analyses, we generated an AtRad52‐1A variant that had a stronger nuclear localization than the native gene thanks to the removal of the transit peptide for mitochondrial localization and to the addition of a nuclear localization signal. Over‐expression of this variant increased HR in the nucleus, compared with the native AtRad52‐1A: it increased intra‐chromosomal recombination and synthesis‐dependent strand‐annealing HR repair rates; but conversely, it repressed the single‐strand annealing pathway. The effect of AtRad52‐1A over‐expression on gene‐targeting was tested with and without the expression of small RNAs generated from an RNAi construct containing homology to the target and donor sequences. True gene‐targeting events at the Arabidopsis Cruciferin locus were obtained only when combining AtRad52‐1A over‐expression and target/donor‐specific RNAi. This suggests that sequence‐specific small RNAs might be involved in AtRad52‐1A‐mediated HR.     

Genome rearrangement in top3 mutants of Saccharomyces cerevisiae requires a functional RAD1 excision repair gene.

Saccharomyces cerevisiae cells that are mutated at TOP3, a gene that encodes a protein homologous to bacterial type I topoisomerases, have a variety of defects, including reduced growth rate, altered gene expression, blocked sporulation, and elevated rates of mitotic recombination at several loci. The rate of ectopic recombination between two unlinked, homologous loci, SAM1 and SAM2, is sixfold higher in cells containing a top3 null mutation than in wild-type cells. Mutations in either of the two other known topoisomerase genes in S. cerevisiae, TOP1 and TOP2, do not affect the rate of recombination between the SAM genes. The top3 mutation also changes the distribution of recombination events between the SAM genes, leading to the appearance of novel deletion-insertion events in which conversion tracts extend beyond the coding sequence, replacing the DNA flanking the 3' end of one SAM gene with nonhomologous DNA flanking the 3' end of the other. The effects of the top3 null mutation on recombination are dependent on the presence of an intact RAD1 excision repair gene, because both the rate of SAM ectopic gene conversion and the conversion tract length were reduced in rad1 top3 mutant cells compared with top3 mutants. These results suggest that a RAD1-dependent function is involved in the processing of damaged DNA that results from the loss of Top3 activity, targeting such DNA for repair by recombination.     

The structure-specific endonuclease Ercc1-Xpf is required for targeted gene replacement in embryonic stem cells.

The Ercc1-Xpf heterodimer, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair and homologous recombination. Ercc1-Xpf incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here we demonstrate that although Ercc1 is dispensable for recombination between sister chromatids, it is essential for targeted gene replacement in mouse embryonic stem cells. Surprisingly, the role of Ercc1-Xpf in gene targeting is distinct from its previously identified role in removing nonhomologous termini from recombination intermediates because it was required irrespective of whether the ends of the DNA targeting constructs were heterologous or homologous to the genomic locus. Our observations have implications for the mechanism of gene targeting in mammalian cells and define a new role for Ercc1-Xpf in mammalian homologous recombination. We propose a model for the mechanism of targeted gene replacement that invokes a role for Ercc1-Xpf in making the recipient genomic locus receptive for gene replacement.     

The Saccharomyces Cerevisiae Ku Autoantigen Homologue Affects Radiosensitivity Only in the Absence of Homologous Recombination

In mammalian cells, all subunits of the DNA-dependent protein kinase (DNA-PK) have been implicated in the repair of DNA double-strand breaks and in V(D)J recombination. In the yeast Saccharomyces cerevisiae, we have examined the phenotype conferred by a deletion of HDF1, the putative homologue of the 70-kD subunit of the DNA-end binding Ku complex of DNA-PK. The yeast gene does not play a role in radiation-induced cell cycle checkpoint arrest in G(1) and G(2) or in hydroxyurea-induced checkpoint arrest in S. In cells competent for homologous recombination, we could not detect any sensitivity to ionizing radiation or to methyl methanesulfonate (MMS) conferred by a hdf1 deletion and indeed, the repair of DNA double-strand breaks was not impaired. However, if homologous recombination was disabled (rad52 mutant background), inactivation of HDF1 results in additional sensitization toward ionizing radiation and MMS. These results give further support to the notion that, in contrast to higher eukaryotic cells, homologous recombination is the favored pathway of double-strand break repair in yeast whereas other competing mechanisms such as the suggested pathway of DNA-PK-dependent direct break rejoining are only of minor importance.     

PSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: probing Nicotiana benthamiana-virus interactions

Plant functional proteomics research is increasingly dependent upon vectors that facilitate high-throughput gene cloning and expression of fusions to autofluorescent proteins. Here, we describe the pSITE family of plasmids, a new set of Agrobacterium binary vectors, suitable for the stable integration or transient expression of various autofluorescent protein fusions in plant cells. The pSITE vectors permit single-step Gateway-mediated recombination cloning for construction of binary vectors that can be used directly in transient expression studies or for the selection of transgenic plants on media containing kanamycin. These vectors can be used to express native proteins or fusions to monmeric red fluorescent protein or the enhanced green fluorescent protein and its cyan and yellow-shifted spectral variants. We have validated the vectors for use in transient expression assays and for the generation of transgenic plants. Additionally, we have generated markers for fluorescent highlighting of actin filaments, chromatin, endoplasmic reticulum, and nucleoli. Finally, we show that pSITE vectors can be used for targeted gene expression in virus-infected cells, which should facilitate high-throughput characterization of protein dynamics in host-virus interactions.     

Targeted disruption of the tissue inhibitor of metalloproteinases gene increases the invasive behavior of primitive mesenchymal cells derived from embryonic stem cells in vitro.

The metalloproteinase family of proteolytic enzymes can degrade extracellular matrix and facilitate invasive migration. This class of enzymes is specifically inhibited by the tissue inhibitor of metalloproteinases (TIMP-1). Using homologous recombination, we have disrupted the gene encoding TIMP-1 in pluripotent embryonic stem cells. Because the TIMP-1 gene is X linked and is hemizygous in embryonic stem cells, we have been able to study the effect of this mutation in culture. Using a basement membrane invasion assay, we found that the mutant cells, differentiated in low concentrations of serum with retinoic acid, were more invasive than their normal cell counterparts, and that this was specifically reversed by adding exogenous TIMP-1 protein. The invasive cell population had characteristics of an early population of primitive mesenchymal cells, including expression of vimentin and a transient period of invasiveness from 4-8 d after initiation of differentiation. Therefore, metalloproteinase activity can be rate limiting for cell invasion.