The structure-specific endonuclease Ercc1-Xpf is required to resolve DNA interstrand cross-link-induced double-strand breaks

Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (gamma-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced gamma-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, gamma-H2AX foci were also induced in Ercc1(-/-) cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1(-/-) cells, MMC-induced gamma-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1(-/-) and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.     

Role of ERCC1 in removal of long non-homologous tails during targeted homologous recombination

The XpF/Ercc1 structure-specific endonuclease performs the 5' incision in nucleotide excision repair and is the apparent mammalian counterpart of the Rad1/Rad10 endonuclease from Saccharomyces cerevisiae. In yeast, Rad1/Rad10 endonuclease also functions in mitotic recombination. To determine whether XpF/Ercc1 endonuclease has a similar role in mitotic recombination, we targeted the APRT locus in Chinese hamster ovary ERCC1(+) and ERCC1(-) cell lines with insertion vectors having long or short terminal non-homologies flanking each side of a double-strand break. No substantial differences were evident in overall recombination frequencies, in contrast to results from targeting experiments in yeast. However, profound differences were observed in types of APRT(+) recombinants recovered from ERCC1(-) cells using targeting vectors with long terminal non-homologies-almost complete ablation of gap repair and single-reciprocal exchange events, and generation of a new class of aberrant insertion/deletion recombinants absent in ERCC1(+) cells. These results represent the first demonstration of a requirement for ERCC1 in targeted homologous recombination in mammalian cells, specifically in removal of long non-homologous tails from invading homologous strands.     

Opposing roles for DNA structure-specific proteins Rad1, Msh2, Msh3, and Sgs1 in yeast gene targeting

Targeted gene replacement (TGR) in yeast and mammalian cells is initiated by the two free ends of the linear targeting molecule, which invade their respective homologous sequences in the chromosome, leading to replacement of the targeted locus with a selectable gene from the targeting DNA. To study the postinvasion steps in recombination, we examined the effects of DNA structure-specific proteins on TGR frequency and heteroduplex DNA formation. In strains deleted of RAD1, MSH2, or MSH3, we find that the frequency of TGR is reduced and the mechanism of TGR is altered while the reverse is true for deletion of SGS1, suggesting that Rad1 and Msh2:Msh3 facilitate TGR while Sgs1 opposes it. The altered mechanism of TGR in the absence of Msh2:Msh3 and Rad1 reveals a separate role for these proteins in suppressing an alternate gene replacement pathway in which incorporation of both homology regions from a single strand of targeting DNA into heteroduplex with the targeted locus creates a mismatch between the selectable gene on the targeting DNA and the targeted gene in the chromosome.     

Double-strand gap repair in a mammalian gene targeting reaction.

To better understand the mechanism of homologous recombination in mammalian cells that facilitates gene targeting, we have analyzed the recombination reaction that inserts a plasmid into a homologous chromosomal locus in mouse embryonic stem cells. A partially deleted HPRT gene was targeted with various plasmids capable of correcting the mutation at this locus, and HPRT+ recombinants were directly selected in HAT medium. The structures of the recombinant loci were then determined by genomic Southern blot hybridizations. We demonstrate that plasmid gaps of 200, 600, and 2,500 bp are efficiently repaired during the integrative recombination reaction. Targeting plasmids that carry a double-strand break or gap in the region of DNA homologous to the target locus produce 33- to 140-fold more hypoxanthine-aminopterin-thymidine-resistant recombinants than did these same plasmids introduced in their uncut (supercoiled) forms. Our data suggest that double-strand gaps and breaks may be enlarged prior to the repair reaction since sequence heterologies carried by the incoming plasmids located close to them are often lost. These results extend the known similarities between mammalian and yeast recombination mechanisms and suggest several features of the insertional (O-type) gene targeting reaction that should be considered when one is designing mammalian gene targeting experiments.     

Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens

In the moss Physcomitrella patens, transforming DNA containing homologous sequences integrates predominantly by homologous recombination with its genomic target. A systematic investigation of the parameters that determine gene targeting efficiency shows a direct relationship between homology length and targeting frequency for replacement vectors (a selectable marker flanked by homologous DNA). Overall homology of only 1 kb is sufficient to achieve a 50% yield of targeted transformants. Targeting may occur through homologous recombination in one arm, accompanied by non-homologous end-joining by the other arm of the vector, or by allele replacement following two homologous recombination events. Allele replacement frequency depends on the symmetry of the targeting vector, being proportional to the length of the shorter arm. Allele replacement may involve insertion of multiple copies of the transforming DNA, accompanied by ectopic insertions at non-homologous sites. Single-copy and single insertions at targeted loci (targeted gene replacements, ‘TGR’) occur with a frequency of 7–20% of all transformants when the minimum requirements for allele replacement are met. Homologous recombination in Physcomitrella is substantially more efficient than in any multicellular eukaryote, recommending it as the outstanding model for the study of homologous recombination in plants.     

Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells.

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.     

基因打靶及其应用

用活细胞染色体DNA可与外源性DNA同源序列发生同源重组的性质,达到定点修饰改造染色体某基因的目的,此法称基因打靶．基因的同源重组是较普遍的生物现象,其分子机理尚未阐明,但活细胞内确有一酶系可使DNA的同源序列在细胞内发生重组,这一事实已无可争辨．此事实为基因打靶的理论基础．基因打靶技术操作的关键是建立一含筛选基因的重组载体,并有效地把它转入细胞核内．基因打靶命中的细胞可稳定遗传．基因打靶在改造生物品种,一些复杂生命现象（如发育的分子机制等）及临床理论研究均有广阔的前景．     

Mechanisms involved in targeted gene replacement in mammalian cells

The "ends-out" or omega (Omega)-form gene replacement vector is used routinely to perform targeted genome modification in a variety of species and has the potential to be an effective vehicle for gene therapy. However, in mammalian cells, the frequency of this reaction is low and the mechanism unknown. Understanding molecular features associated with gene replacement is important and may lead to an increase in the efficiency of the process. In this study, we investigated gene replacement in mammalian cells using a powerful assay system that permits efficient recovery of the product(s) of individual recombination events at the haploid, chromosomal mu-delta locus in a murine hybridoma cell line. The results showed that (i) heteroduplex DNA (hDNA) is formed during mammalian gene replacement; (ii) mismatches in hDNA are usually efficiently repaired before DNA replication and cell division; (iii) the gene replacement reaction occurs with fidelity; (iv) the presence of multiple markers in one homologous flanking arm in the replacement vector did not affect the efficiency of gene replacement; and (v) in comparison to a genomic fragment bearing contiguous homology to the chromosomal target, gene targeting was only slightly inhibited by internal heterology (pSV2neo sequences) in the replacement vector.     

End extension repair of introduced targeting vectors mediated by homologous recombination in mammalian cells.

We have studied the mechanism of targeted recombination in mammalian cells using a hemizygous adenine phosphoribosyltransferase-deficient (APRT-) Chinese hamster ovary (CHO) cell mutant as a recipient. Three structurally different targeting vectors with a 5' or a 3', or both, end-deleted aprt sequence, in either a closed-circular or linear form, were transfected to the cells with a mutated aprt gene by electroporation. APRT-positive (APRT+) recombinant clones were selected and analyzed to study the gene correction events of the deletion mutation. Some half of 58 recombinant clones obtained resulted from corrections of the deleted chromosomal aprt gene by either gene replacement or gene insertion, a mechanism which is currently accepted for homologous recombination in mammalian cells. However, the chromosomal sequence in the remaining half of the recombinants remained uncorrected but their truncated end of the aprt gene in the incoming vectors was corrected by extending the end beyond the region of homology to the target locus; the corrected vector was then randomly integrated into the genome. This extension, termed end extension repair, was observed with all three vectors used and was as far as 4.6-kilobase (kb) or more long. It is evident that the novel repair reaction mediated by homologous recombination, in addition to gene replacement and gene insertion, is also involved in gene correction events in mammalian cells. We discuss the model which may account for this phenomenon.     

PCR-based gene targeting of the inducible nitric oxide synthase (NOS2) locus in murine ES cells,a new and more cost-effective approach

Gene targeting by double homologous recombination in murine embryonic stem (ES) cells is a powerful tool used to study the cellular consequences of specific genetic mutations. A typical targeting construct consists of a neomycin phosphotransferase (neo) gene flanked by genomic DNA fragments that are homologous to sequences in the target chromosomal locus. Homologous DNA fragments are typically cloned from a murine genomic DNA library. Here we describe an alternative approach whereby the inducible nitric oxide synthase (NOS2) gene locus is partially mapped and homologous DNA sequences obtained using a long-range PCR method. A 7 kb NOS2 amplicon is used to construct a targeting vector where theneo gene is flanked by PCR-derived homologous DNA sequences. The vector also includes a thymidine kinase (tk) negative-selectable marker gene. Following transfection into ES cells, the PCR-based targeting vector undergoes efficient homologous recombination into the NOS2 locus. Thus, PCR-based gene targeting can be a valuable alternative to the conventional cloning approach. It expedites the acquisition of homologous genomic DNA sequences and simplifies the construction of targeting plasmids by making use of defined cloning sites. This approach should result in substantial time and cost savings for appropriate homologous recombination projects.     

Replacement-like recombination induced by an integration vector with a murine homology flank at the immunoglobulin heavy-chain locus in mouse and rat hybridoma cells

Vectors for homologous recombination are commonly designed as replacement or integration constructs. We have evaluated integration vectors for the substitution of the immunoglobulin heavy-chain constant region by various human isotypes in mouse and rat hybridomas. It is known that under certain circumstances replacement vectors exhibit a lower target efficiency and can be incorporated by integration events. Conversely, we show here that an integration vector can undergo a replacement event despite having free homologous adjacent DNA ends, which would be expected to initiate integration according to the double-strand break repair model. Moreover, in cases of replacement recombination the 5 crossover is not necessarily located within the homology region, thereby giving rise to a truncated gene product. Whether or not the replacement leads to such deletions is clearly dependent on the isotypes involved in the targeting reaction. The fact that the vector is correctly targeted to the heavy-chain locus, but that the homology region is not always the site of recombination, points to a novel recombination mechanism that may be specific for the immunoglobulin loci and that seems to be predominant even in the presence of the free homologous adjacent ends of an integration vector. Furthermore we demonstrate that homologous recombination at the heavy-chain locus is also possible between sequences from different species. The implications of our findings for the production of chimeric antibodies are discussed.     

Parameters controlling the rate of gene targeting frequency in the protozoan parasite Leishmania.

In this study we investigated the role of several parameters governing the efficiency of gene targeting mediated by homologous recombination in the protozoan parasite Leishmania. We evaluated the relative targeting frequencies of different replacement vectors designed to target several sequences within the parasite genome. We found that a decrease in the length of homologous sequences <1 kb on one arm of the vector linearly influences the targeting frequency. No homologous recombination was detected, however, when the flanking homologous regions were <180 bp. A requirement for a very high degree of homology between donor and target sequences was found necessary for efficient gene targeting in Leishmania , as targeted recombination was strongly affected by base pair mismatches. Targeting frequency increased proportionally with copy number of the target only when the target was part of a linear amplicon, but remained unchanged when it was present on circles. Different chromosomal locations were found to be targeted with significantly variable levels of efficiency. Finally, different strains of the same species showed differences in gene targeting frequency. Overall, gene targeting mediated by homologous recombination in Leishmania shares similarities to both the yeast and the mammalian recombination systems.     

Increased Gene Targeting in Ku70 and Xrcc4 Transiently Deficient Human Somatic Cells

The insertion of foreign DNA at a specific genomic locus directed by homologous DNA sequences, or gene targeting, is an inefficient process in mammalian somatic cells. Given the key role of non-homologous end joining (NHEJ) pathway in DNA double-strand break (DSB) repair in mammalian cells, we investigated the effects of decreasing NHEJ protein levels on gene targeting. Here we demonstrate that the transient knockdown of integral NHEJ proteins, Ku70 and Xrcc4, by RNAi in human HCT116 cells has a remarkable effect on gene targeting/random insertions ratios. A timely transfection of an HPRT-based targeting vector after RNAi treatment led to a 70% reduction in random integration events and a 33-fold increase in gene targeting at the HPRT locus. These findings bolster the role of NHEJ proteins in foreign DNA integration in vivo, and demonstrate that their transient depletion by RNAi is a viable approach to increase the frequency of gene targeting events. Understanding how foreign DNA integrates into a cell’s genome is important to advance strategies for biotechnology and genetic medicine.     

Gene targeting in plants: fingers on the move

Zinc-finger endonucleases (ZFNs) make targeted double-stranded breaks in genomic DNA and, thus, stimulate recombination and repair processes at specific sites. ZFNs can now be harnessed to stimulate homologous recombination and gene targeting in plants, which represents a major step towards modifying the plant genome more precisely. ZFN-mediated gene targeting is likely to become a powerful tool for genome research and genetic engineering.     

Involvement of mitochondrial-targeted RecA in the repair of mitochondrial DNA in the moss, Physcomitrella patens

Homologous recombination is a universal process that contributes to genetic diversity and genomic integrity. Bacterial-type RecA generally exists in all bacteria and plays a crucial role in homologous recombination. Although RecA homologues also exist in plant mitochondria, there have been few reports about the in vivo functions of these homologues. We identified a recA gene orthologue (named PprecA1) in a cDNA library of the moss, Physcomitrella patens. N-terminal fusion of the putative organellar targeting sequence of PpRecA1 to GFP caused a targeting of PpRecA1 to mitochondria. PprecA1 partially complemented the effects of a DNA damaging agent in an Escherichia coli recA deficient strain. Additionally, the expression of PprecA1 was induced by treating the plants with DNA damaging agents. Disruption of PprecA1 by targeted replacement resulted lower rate of the recovery of the mitochondrial DNA from methyl methan sulfonate damage. This is the first report about the characteristics of a null mutant of bacterial-type recA gene in plant. The data suggest that PprecA1 participates in the repair of mitochondrial DNA in P. patens.     

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.     

Multiple pathways for repair of DNA double-strand breaks in mammalian chromosomes

To study repair of DNA double-strand breaks (DSBs) in mammalian chromosomes, we designed DNA substrates containing a thymidine kinase (TK) gene disrupted by the 18-bp recognition site for yeast endonuclease I-SceI. Some substrates also contained a second defective TK gene sequence to serve as a genetic donor in recombinational repair. A genomic DSB was induced by introducing endonuclease I-SceI into cells containing a stably integrated DNA substrate. DSB repair was monitored by selection for TK-positive segregants. We observed that intrachromosomal DSB repair is accomplished with nearly equal efficiencies in either the presence or absence of a homologous donor sequence. DSB repair is achieved by nonhomologous end-joining or homologous recombination, but rarely by nonconservative single-strand annealing. Repair of a chromosomal DSB by homologous recombination occurs mainly by gene conversion and appears to require a donor sequence greater than a few hundred base pairs in length. Nonhomologous end-joining events typically involve loss of very few nucleotides, and some events are associated with gene amplification at the repaired locus. Additional studies revealed that precise religation of DNA ends with no other concomitant sequence alteration is a viable mode for repair of DSBs in a mammalian genome.     

Aptamer-guided gene targeting in yeast and human cells

Gene targeting is a genetic technique to modify an endogenous DNA sequence in its genomic location via homologous recombination (HR) and is useful both for functional analysis and gene therapy applications. HR is inefficient in most organisms and cell types, including mammalian cells, often limiting the effectiveness of gene targeting. Therefore, increasing HR efficiency remains a major challenge to DNA editing. Here, we present a new concept for gene correction based on the development of DNA aptamers capable of binding to a site-specific DNA binding protein to facilitate the exchange of homologous genetic information between a donor molecule and the desired target locus (aptamer-guided gene targeting). We selected DNA aptamers to the I-SceI endonuclease. Bifunctional oligonucleotides containing an I-SceI aptamer sequence were designed as part of a longer single-stranded DNA molecule that contained a region with homology to repair an I-SceI generated double-strand break and correct a disrupted gene. The I-SceI aptamer-containing oligonucleotides stimulated gene targeting up to 32-fold in yeast Saccharomyces cerevisiae and up to 16-fold in human cells. This work provides a novel concept and research direction to increase gene targeting efficiency and lays the groundwork for future studies using aptamers for gene targeting.     

Recycling selectable markers in mouse embryonic stem cells.

As a result of gene targeting, selectable markers are usually permanently introduced into the mammalian genome. Multiple gene targeting events in the same cell line can therefore exhaust the pool of markers available and limit subsequent manipulations or genetic analysis. In this study, we describe the combined use of homologous and CRE-loxP-mediated recombination to generate mouse embryonic stem cell lines carrying up to four targeted mutations and devoid of exogenous selectable markers. A cassette that contains both positive and negative selectable markers flanked by loxP sites, rendering it excisable by the CRE protein, was constructed. Homologous recombination and positive selection were used to disrupt the Rep-3 locus, a gene homologous to members of the mutS family of DNA mismatch repair genes. CRE-loxP-mediated recombination and negative selection were then used to recover clones in which the cassette had been excised. The remaining allele of Rep-3 was then subjected to a second round of targeting and excision with the same construct to generate homozygous, marker-free cell lines. Subsequently, both alleles of mMsh2, another mutS homolog, were disrupted in the same fashion to obtain cell lines homozygous for targeted mutations at both the Rep-3 and mMsh2 loci and devoid of selectable markers. Thus, embryonic stem cell lines obtained in this fashion are suitable for further manipulation and analysis involving the use of selectable markers.