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Results for “Homologous Recombination”

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PubMed1998

A partial hprt gene duplication generated by non-homologous recombination in V79 Chinese hamster cells is eliminated by homologous recombination.

Here, the sequence in the hprt gene of the duplication mutant SPD8 originating from V79 Chinese hamster cells was determined. The duplication arose after non-homologous recombination between exon 6 and intron 7, resulting in an extra copy of the 3' portion of exon 6, of exon 7 and of flanking intron regions. Only a duplication of exon 7 is present in the mRNA, since the duplicated exon 6 lacks its 5' splice site and is removed during RNA processing. The findings in this study suggest that the non-homologous recombination mechanism which occurred here may have been initiated by endonucleases, rather than by a spontaneous double strand break. Subsequently, 14 spontaneous SPD8 revertants with a functional hprt gene were isolated and characterized using PCR and sequencing. The data revealed that although the SPD8 cell line arose by non-homologous recombination, it reverts spontaneously by homologous recombination. Interestingly, the downstream copy of exon 7 was restored by this process. This was indicated by the presence of a specific mutation, a T-to-G transversion, close to the breakpoint, a characteristic unique to the SPD8 clone. Our results suggest that the spontaneous reversion of this cell line by homologous recombination may involve an exchange, rather than a conversion mechanism.

Animals↗

PubMed2025

Construction and Isolation of Recombinant Vaccinia Virus by Homologous Recombination Using Fluorescent Protein Markers.

Genetic modification of vaccinia virus (VACV) is a fundamental and valuable research technique in elucidating the function of VACV genes, as well as the development as vaccine vectors for other infectious diseases, oncolytic therapeutics for cancers, and protein expression systems in mammalian cells. Because of the large size of poxvirus genome and noninfectious feature of the naked viral DNA, construction of recombinant VACV relies on intracellular homologous recombination between transfected DNA and replicating viral DNA in infected cells occurred in VACV infected cells. The efficiency of homologous recombination event for vaccinia virus is relatively low, and recombinant viruses only account for 0.1% of progeny viruses. Therefore, fluorescent protein markers are often included in the transfected DNA to facilitate the selection and screening of recombined viruses. Here we provide a detailed procedure for the design, generation, isolation, and detection of recombinant VACV by homologous recombination using fluorescent protein markers.

Vaccinia virus↗

PubMed2005

Comparative and evolutionary analysis of the bacterial homologous recombination systems.

Homologous recombination is a housekeeping process involved in the maintenance of chromosome integrity and generation of genetic variability. Although detailed biochemical studies have described the mechanism of action of its components in model organisms, there is no recent extensive assessment of this knowledge, using comparative genomics and taking advantage of available experimental data on recombination. Using comparative genomics, we assessed the diversity of recombination processes among bacteria, and simulations suggest that we missed very few homologs. The work included the identification of orthologs and the analysis of their evolutionary history and genomic context. Some genes, for proteins such as RecA, the resolvases, and RecR, were found to be nearly ubiquitous, suggesting that the large majority of bacterial genomes are capable of homologous recombination. Yet many genomes show incomplete sets of presynaptic systems, with RecFOR being more frequent than RecBCD/AddAB. There is a significant pattern of co-occurrence between these systems and antirecombinant proteins such as the ones of mismatch repair and SbcB, but no significant association with nonhomologous end joining, which seems rare in bacteria. Surprisingly, a large number of genomes in which homologous recombination has been reported lack many of the enzymes involved in the presynaptic systems. The lack of obvious correlation between the presence of characterized presynaptic genes and experimental data on the frequency of recombination suggests the existence of still-unknown presynaptic mechanisms in bacteria. It also indicates that, at the moment, the assessment of the intrinsic stability or recombination isolation of bacteria in most cases cannot be inferred from the identification of known recombination proteins in the genomes.

Journal Article↗

PubMed1995

Dependence of frequency of homologous recombination on the homology length.

The frequency of homologous recombination is believed to be a linear function of the length (N bp) of homology between DNAs. Here, the N intercept is believed to be determined by a threshold length below which some physical constraint is effective. In the mammalian gene targeting systems, however, the frequency depends more steeply than linearly on the homology length. To explain both the linear dependence and the steeper dependence, we propose a model where the branch point of a reaction intermediate is assumed to "walk randomly" along the homologous region until it is processed. The intermediate is assumed to be destroyed if the branch point ever reaches either end of the homology. In this model, the length dependence is governed by a parameter, h, which is defined as efficiency of processing of the intermediate and reflects unlikelihood of the destruction at either end of the homology. We find that the frequency is proportional to N3 for smaller N and is a linear function of N for larger N. Where the shift from the N3 dependence to the linear dependence takes place is determined by the parameter h. The range of N showing the N3 dependence becomes narrower as h becomes larger. The dependence steeper than linear dependence, which is observed not only in the mammalian gene targeting system but also in bacteriophage T4, Escherichia coli and yeast systems, agrees well with the predicted N3 dependence. The N intercept is determined not by physical (or structural) constraints but only by the parameter h in this model.

Animals↗

PubMed1996

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.

Base Sequence↗

PubMed1999

BRCA1 deficient embryonic stem cells display a decreased homologous recombination frequency and an increased frequency of non-homologous recombination that is corrected by expression of a brca1 transgene.

BRCA1 is a nuclear phosphoprotein that has been classified as a tumor suppressor based on the fact that women carrying a mutated copy of the BRCA1 gene are at increased risk of developing breast and ovarian cancer. The association of BRCA1 with RAD51 has led to the hypothesis that BRCA1 is involved in DNA repair. We describe here the generation and analysis of murine embryonic stem (ES) cell lines in which both copies of the murine homologue of the human BRCA1 gene have been disrupted by gene targeting. We show that exogenous DNA introduced into these BRCA1 deficient cells by electroporation is randomly integrated into the genome at a significantly higher rate than in wild type ES cells. In contrast, integration of exogenous DNA by homologous recombination occurs in BRCA1 deficient cells at a significantly lower rate than in wild type controls. When BRCA1 expression is re-established at 5-10% of normal levels by introduction of a Brca1 transgene into BRCA1 deficient ES cells, the frequency of random integration is reduced to wild type levels, although the frequency of homologous recombination is not significantly improved. These results suggest that BRCA1 plays a role in determining the response of cells to double stranded DNA breaks.

Animals↗

PubMed2001

A novel meiosis-specific protein of fission yeast, Meu13p, promotes homologous pairing independently of homologous recombination.

Meiotic homologous pairing is crucial to proper homologous recombination, which secures subsequent reductional chromosome segregation. We have identified a novel meiosis-specific protein of fission yeast Schizosaccharomyces pombe, Meu13p, to be a molecule that is required for proper homologous pairing and recombination. Rec12p (homologue of Saccharomyces cerevisiae Spo11p), which is essential for the initiation of meiotic recombination, is also shown for the first time to participate in the pairing process of S.pombe. Meu13p, however, contributes to pairing through a recombination-independent mechanism, as disruption of the meu13(+) gene reduces pairing whether the rec12(+) gene is deleted or not. We also demonstrate a dynamic nature of homologous pairing in living meiotic cells, which is markedly affected by meu13 deletion. Meu13p is not required for telomere clustering and the nuclear movement process, which are well known requirements for efficient pairing in S.pombe. Based on these results, together with the localization of Meu13p on meiotic chromatin, we propose that Meu13p directly promotes proper homologous pairing and recombination.

Amino Acid Sequence↗

PubMed1987

Modification of DNA ends can decrease end joining relative to homologous recombination in mammalian cells.

In animal cells, exogenous DNA recombines into random chromosomal sites much more frequently than it recombines into homologous sites. Free DNA ends are "recombinogenic" in both processes. To test the effects of specific ends on analogous extrachromosomal processes, we constructed a linear genome of simian virus 40 with terminal repeated sequences. After transfection into monkey cells, the model substrate can circularize by end joining (analogous to random integration) or by homologous recombination between its terminal repeats (analogous to targeted recombination). Since the two types of recombination are in competition with one another, the ratio of homologous-recombination to end-join products is a sensitive indicator of the differential effects of specific ends. Substrates with blunt ends, complementary sticky ends, or mismatched ends generated the same ratio of homologous-recombination to end-join products. However, addition of dideoxynucleotides to the 3' hydroxyls of the substrate decreased the frequency of end joining by a factor of 5-6 relative to homologous recombination. Thus, the frequency of end joining can be decreased relative to that of homologous recombination by modification of the ends of the input DNA. These results suggest an approach to altering the ratio of random to targeted integration in mammalian cells.

Animals↗

PubMed2005

Multifaceted role of the Saccharomyces cerevisiae Srs2 helicase in homologous recombination regulation.

Homologous recombination (HR) is a major pathway for the elimination of DNA DSBs (double-strand breaks) induced by high-energy radiation and chemicals, or that arise due to endogenous damage and stalled DNA replication forks. If not processed properly, DSBs can lead to cell death, chromosome aberrations and tumorigenesis. Even though HR is important for genome maintenance, it can also interfere with other DNA repair mechanisms and cause gross chromosome rearrangements. In addition, HR can generate DNA or nucleoprotein intermediates that elicit prolonged cell-cycle arrest and sometimes cell death. Genetic analyses in the yeast Saccharomyces cerevisiae have revealed a central role of the Srs2 helicase in preventing untimely HR events and in inhibiting the formation of potentially deleterious DNA structures or nucleoprotein complexes upon DNA replication stress. Paradoxically, efficient repair of DNA DSBs by HR is dependent on Srs2. In this paper, we review recent molecular studies aimed at deciphering the multifaceted role of Srs2 in HR and other cellular processes. These studies have provided critical insights into how HR is regulated in order to preserve genomic integrity and promote cell survival.

Cytoskeleton↗

PubMed2005

The BRCA2-interacting protein BCCIP functions in RAD51 and BRCA2 focus formation and homologous recombinational repair.

Homologous recombinational repair (HRR) of DNA damage is critical for maintaining genome stability and tumor suppression. RAD51 and BRCA2 colocalization in nuclear foci is a hallmark of HRR. BRCA2 has important roles in RAD51 focus formation and HRR of DNA double-strand breaks (DSBs). We previously reported that BCCIPalpha interacts with BRCA2. We show that a second isoform, BCCIPbeta, also interacts with BRCA2 and that this interaction occurs in a region shared by BCCIPalpha and BCCIPbeta. We further show that chromatin-bound BRCA2 colocalizes with BCCIP nuclear foci and that most radiation-induced RAD51 foci colocalize with BCCIP. Reducing BCCIPalpha by 90% or BCCIPbeta by 50% by RNA interference markedly reduces RAD51 and BRCA2 foci and reduces HRR of DSBs by 20- to 100-fold. Similarly, reducing BRCA2 by 50% reduces RAD51 and BCCIP foci. These data indicate that BCCIP is critical for BRCA2- and RAD51-dependent responses to DNA damage and HRR.

BRCA2 Protein↗

PubMed1998

Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells.

Eukaryotic cells repair DNA double-strand breaks (DSBs) by at least two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad54 participates in the first recombinational repair pathway while Ku proteins are involved in NHEJ. To investigate the distinctive as well as redundant roles of these two repair pathways, we analyzed the mutants RAD54(-/-), KU70(-/-) and RAD54(-/-)/KU70(-/-), generated from the chicken B-cell line DT40. We found that the NHEJ pathway plays a dominant role in repairing gamma-radiation-induced DSBs during G1-early S phase while recombinational repair is preferentially used in late S-G2 phase. RAD54(-/-)/KU70(-/-) cells were profoundly more sensitive to gamma-rays than either single mutant, indicating that the two repair pathways are complementary. Spontaneous chromosomal aberrations and cell death were observed in both RAD54(-/-) and RAD54(-/-)/KU70(-/-) cells, with RAD54(-/-)/KU70(-/-) cells exhibiting significantly higher levels of chromosomal aberrations than RAD54(-/-) cells. These observations provide the first genetic evidence that both repair pathways play a role in maintaining chromosomal DNA during the cell cycle.

Amino Acid Sequence↗

PubMed2006

Relative contribution of homologous recombination and non-homologous end-joining to DNA double-strand break repair after oxidative stress in Saccharomyces cerevisiae.

Oxidative damage to DNA seems to be an important factor in developing many human diseases including cancer. It involves base and sugar damage, base-free sites, DNA-protein cross-links and DNA single-strand (SSB) and double-strand (DSB) breaks. Oxidative DSB can be formed in various ways such as their direct induction by the drug or their generation either through attempted and aborted repair of primary DNA lesions or through DNA replication-dependent conversion of SSB. In general, two main pathways are responsible for repairing DSB, homologous recombination (HR) and non-homologous end-joining (NHEJ), with both of them being potential candidates for the repair of oxidative DSB. We have examined relative contribution of HR and NHEJ to cellular response after oxidative stress in Saccharomyces cerevisiae. Therefore, cell survival, mutagenesis and DSB induction and repair in the rad52, yku70 and rad52 yku70 mutants after hydrogen peroxide (H(2)O(2)), menadione (MD) or bleomycin (BLM) exposure were compared to those obtained for the corresponding wild type. We show that MD exposure does not lead to observable DSB induction in yeast, suggesting that the toxic effects of this agent are mediated by other types of DNA damage. Although H(2)O(2) treatment generates some DSB, their yield is relatively low and hence DSB may only partially be responsible for toxicity of H(2)O(2), particularly at high doses of the agent. On the other hand, the basis of the BLM toxicity resides primarily in DSB induction. Both HR and NHEJ act on BLM-induced DSB, although their relative participation in the process is not equal. Based on our results we suggest that the complexity and/or the quality of the BLM-induced DSB might represent an obstacle for the NHEJ pathway.

Bleomycin↗

PubMed2004

Uncommon deletions of the Smith-Magenis syndrome region can be recurrent when alternate low-copy repeats act as homologous recombination substrates.

Several homologous recombination "hotspots," or sites of positional preference for strand exchanges, associated with recurrent deletions and duplications have been reported within large low-copy repeats (LCRs). Recently, such a hotspot was identified in patients with the Smith-Magenis syndrome (SMS) common deletion of approximately 4 Mb or a reciprocal duplication within the KER gene cluster of the SMS-REP LCRs, in which 50% of analyzed strand exchanges resulting in deletion and 23% of those resulting in duplication occurred. Here, we report an additional recombination hotspot within LCR17pA and LCR17pD, which serve as alternative substrates for nonallelic homologous recombination that results in large (approximately 5 Mb) deletions of 17p11.2, which include the SMS region. Using polymerase-chain-reaction mapping of somatic cell hybrid lines, we refined the breakpoints of six deletions within these LCRs. Sequence analysis of the recombinant junctions revealed that all six strand exchanges occurred within a 524-bp interval, and four of them occurred within an AluSq/x element. This interval represents only 0.5% of the 124-kb stretch of 98.6% sequence identity between LCR17pA and LCR17pD. A search for potentially stimulating sequence motifs revealed short AT-rich segments flanking the recombination hotspot. Our findings indicate that alternative LCRs can mediate rearrangements, resulting in haploinsufficiency of the SMS critical region, and reimplicate homologous recombination as a major mechanism for genomic disorders.

Abnormalities, Multiple↗

PubMed2005

Cisplatin sensitization by concurrent mild hyperthermia in parental and mutant cell lines deficient in homologous recombination and non-homologous endjoining repair.

The effect of mild hyperthermia on cisplatin sensitization was examined in two cell line pairs, CHO parental AA8 and irsISF, an XRCC3 mutant (deficient in homologous recombination repair), and mouse parental MEF and knockout Ku80 mutants (deficient in non-homologous endjoining repair). The results showed that mild hyperthermia 40, 41 and 42 degrees C given concurrently with cisplatin treatment caused significant sensitization. The degree of sensitization was comparable for the parental and mutant lines, indicating that these repair pathways were likely not involved in cisplatin thermal sensitization. The shorter concurrent treatments cause a larger sensitization than the longer treatments. The reasons for this are not clear, but thermotolerance may be a factor.

Animals↗

PubMed1996

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.

Adenoviruses, Human↗

PubMed2003

Interactive competition between homologous recombination and non-homologous end joining.

DNA-dependent protein kinase (DNA-PK), composed of Ku70, Ku80, and the catalytic subunit (DNA-PKcs), is involved in double-strand break (DSB) repair by non-homologous end joining (NHEJ). DNA-PKcs defects confer ionizing radiation sensitivity and increase homologous recombination (HR). Increased HR is consistent with passive shunting of DSBs from NHEJ to HR. We therefore predicted that inhibiting the DNA-PKcs kinase would increase HR. A novel DNA-PKcs inhibitor (1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone; designated IC86621) increased ionizing radiation sensitivity but surprisingly decreased spontaneous and DSB-induced HR. Wortmannin also inhibits DNA-PKcs and reduces DSB-induced HR. IC86621 did not affect HR product outcome, indicating that it affects HR initiation. Thus, HR is increased in the absence of DNA-PKcs, but decreased when DNA-PKcs is catalytically inactive, suggesting interactive competition between HR and NHEJ. The effects of IC86621 and wortmannin were proportional to the level of DNA-PKcs, consistent with inhibited DNA-PKcs acting in a dominant negative manner. We propose that inhibition of DNA-PKcs blocks its autophosphorylation, prevents dissociation of DNA-PKcs from DNA ends, and thereby blocks both HR and NHEJ. By blocking the two major DSB repair pathways, DNA-PKcs inhibitors should radiosensitize at all cell-cycle stages and are therefore excellent candidates for augmenting cancer radiotherapy.

Acetophenones↗

PubMed1994

DNA replication triggered by double-stranded breaks in E. coli: dependence on homologous recombination functions.

Homologous recombination-dependent DNA replication (RDR) of a lambda cos site-carrying plasmid is demonstrated in E. coli cells when the cells express lambda terminase that introduces a double-stranded break into the cos site. RDR occurs in normal wild-type cells if the plasmid also contains the recombination hotspot chi. Chi is dispensable when cells are induced for the SOS response or contain a recD mutation. recBC sbcA mutant cells are also capable of RDR induction. A recN mutation greatly reduces RDR in normal cells, but not in SOS-induced cells. RDR proceeds by the theta mode or rolling circle mode of DNA synthesis, yielding covalently closed circular plasmid monomers or linear plasmid multimers, respectively. Previously described inducible stable DNA replication is considered to be a special type of RDR that starts exclusively from specific sites (oriMs) on the chromosome.

Bacterial Proteins↗

PubMed1994

Production of mouse V/human C chimeric kappa genes by homologous recombination in hybridoma cells. Analysis of vector design and recombinant gene expression.

Homologous recombination between transferred and chromosomal Ig genes in mouse hybridoma cells offers a general method of altering the chromosomal Ig genes in predetermined ways. Recombination is infrequent in hybridoma cells, and we have been interested in improving the methods for identifying and recovering the rare recombinants. We have used vectors that are designed to replace the mouse chromosomal C kappa segment with the human equivalent, so that recombinants produce mouse V/human C chimeric kappa-chains. We describe an enhancerless, replacement type vector that can be used with the herpes thymidine kinase counterselection to provide such enrichment that homologous recombinants constitute 15% of the selected G418-resistant, FIAU-resistant cells. We have also measured the level of chimeric kappa gene expression and found surprisingly that (1) it is very variable among transformants with the same recombinant gene structure, (2) there is no systematic difference in the level of production by recombinants that retain or have lost the J-C kappa intron enhancer, and (3) the amount of chimeric kappa mRNA in even the highest producing transformants is much less than the amount of the corresponding mouse kappa mRNA.

Animals↗