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PubMed2003

Activities of wildtype and mutant p53 in suppression of homologous recombination as measured by a retroviral vector system.

DNA repair of double strand breaks, interstrand DNA cross-links, and other types of DNA damage utilizes the processes of homologous recombination and non-homologous end joining to repair the damage. Aberrant homologous recombination is likely to be responsible for a significant fraction of chromosomal deletions, duplications, and translocations that are observed in cancer cells. To facilitate measurement of homologous recombination frequencies in normal cells, mutant cells, and cancer cells, we have developed a high titer retroviral vector containing tandem repeats of mutant versions of a GFP-Zeocin resistance fusion gene and an intact neomycin resistance marker. Recombination between the tandem repeats regenerates a functional GFP-Zeo(R) marker that can be easily scored. This retroviral vector was used to assess homologous recombination frequencies in human cancer cells and rodent fibroblasts with differing dosages of wild type or mutant p53. Absence of wild type p53 stimulated spontaneous and ionizing radiation-induced homologous recombination, confirming previous studies. Moreover, p53(+/-) mouse fibroblasts show elevated levels of homologous recombination compared to their p53(+/+) counterparts following retroviral vector infection, indicating that p53 is haploinsufficient for suppression of homologous recombination. Transfection of vector-containing p53 null Saos-2 cells with various human cancer-associated p53 mutants revealed that these altered p53 proteins retain some recombination suppression function despite being totally inactive for transcriptional transactivation. The retroviral vector utilized in these studies may be useful in performing recombination assays on a wide array of cell types, including those not readily transfected by normal vectors.

DNA Repair↗
PubMed2002

Construction of double-copy glucose isomerase gene engineering strain of Streptomyces diastaticus by homologous recombination.

The plasmid pUT for homologous recombination was constructed by the insertion of the 1.1-kb thiostrepton resistance ( tsr(R)) gene into the E. coli plasmid pUB1-GI1. Plasmid pUTK was produced through ligating the cleaved plasmid pUT by KpnI. After pUT and pUTK were introduced into Streptomyces diastaticus No.7 strain M1033 (SM33) by protoplast transformation, a series of tsr(R) transformants were obtained, further based on enzyme assays. These results for polymerase chain reaction (PCR), DNA sequencing, restriction enzyme digestion, and recovery of cloned fragments from the transformant chromosome demonstrated the plasmid pUT and pUTK had integrated into the SM33 chromosome in three different patterns of single cross-over by homologous recombination. This directly results in double-copy GI gene in the transformant chromosome, of which one is wild-type GI gene, the other mutant GI ( GIG138P, GI1) gene. Among the strains of the three kinds of recombinant patterns, one transformant was chosen and named K1, T2, and T3, respectively. The further identification of the three recombinant strains by PCR, DNA sequencing, restriction enzyme digestion, and Southern hybridization also proved there is a double-copy GI gene within their chromosome. Enzyme activity assay and thermostability analysis indicated that all three engineering strains expressed not only wild-type enzyme but also mutant GI.

Aldose-Ketose Isomerases↗
PubMed2001

[Homologous recombination and chromosomal rearrangements in Escherichia coli strains carrying a heterozygous tandem duplication].

Heterozygous tandem duplications formed in conjugational matings in Escherichia coli provides a convenient model system for studying the evolution of bacterial chromosome. Heterozygous duplications segregate various classes of haploid and diploid recombinants that appear as a result of unequal crossing over between sister chromosomes. In this work, an extended tandem duplication in the deo operon of E. coli carrying deoA deoB::Tn5/deoC deoD thr::Tn9 alleles was examined. Recombination between homologous DNA repeats in the duplication was studied in strains carrying different combinations of recBC, sbcBC, recB::Tn10, recQ::Tn3 mutations. The frequency of recombination between homologous DNA repeats was very high in all strains and did not decrease when the RecBCD and RecF recombinational pathways were simultaneously damaged in strains with the recB sbcBC recQ (or recF) genotype. It is assumed that unequal crossing over between direct DNA repeats in duplications may proceed through a particular pathway of "adaptive" recombination.

Alleles↗
PubMed1989

High-frequency homologous recombination between duplicate chromosomal immunoglobulin mu heavy-chain constant regions.

Homologous recombination was used in a previous study to correct a 2-base-pair deletion in the third constant domain (Cmu3) of the haploid chromosomal mu gene in a mutant hybridoma cell line by transfer of a pSV2neo vector bearing a subfragment of the normal Cmu region (M.D. Baker, N. Pennell, L. Bosnoyan, and M.J. Shulman, Proc. Natl. Acad. Sci. USA 85:6432-6436, 1988). In these experiments, both gene replacement and single reciprocal crossover events were found to restore normal, cytolytic 2,4,6-trinitrophenyl-specific immunoglobulin M production to the mutant cells. In the cases of single reciprocal recombination, the structure of the recombinant mu gene is such that the normal Cmu region, in its correct position 3' of the expressed 2,4,6-trinitrophenyl-specific heavy-chain variable region, is separated from the mutant Cmu region by the integrated vector sequences. I report here that homologous recombination occurs with high frequency between the duplicate Cmu regions in mitotically growing hybridoma cells. The homologous recombination events were easily detected since they generated hybridomas that were phenotypically different from the parental cells. Analysis of the recombinant cells suggests that gene conversion is the most frequent event, occurring between 60 and 73% of the time. The remaining events consisted of single reciprocal crossovers. Intrachromatid double reciprocal recombination was not detected. The high frequency of recombination, the ability to isolate and analyze the participants in the recombination reactions, and the capacity to generate specific modifications in the immunoglobulin Cmu regions by gene targeting suggest that this system will be useful for studying mammalian chromosomal homologous recombination. Moreover, the ability to specifically modify the chromosomal immunoglobulin genes by homologous recombination should facilitate studies of immunoglobulin gene regulation and expression and provide a more convenient of engineering specifically modified antibody.

Animals↗
PubMed2001

Distinctive features of homologous recombination in an 'old' microorganism, Lactococcus lactis.

Homologous recombination is needed to assure faithful inheritance of DNA material, especially under stress conditions. The same enzymes that repair broken chromosomes via recombination also generate biodiversity. Their activities may result in intrachromosomal rearrangements, assimilation of foreign DNA, or a combination of these events. It is generally supposed that homologous recombination systems are conserved, and function the same way everywhere as they do in Escherichia coli, the accepted paradigm. Studies in an 'older' microorganism, the gram-positive bacterium of the low GC branch Lactococcus lactis, confirm that many enzymes are conserved across species lines. However, the main components of the double strand break (DSB) repair system, an exonuclease/helicase (Exo/hel) and a short DNA modulator sequence Chi, differ markedly between bacteria, especially when compared to the gram-negative analogues. Based on our studies, a model is proposed for the functioning of the two-subunit Exo/hel of L. lactis and other gram-positive bacteria, which differs from that of the three-subunit E. coli enzyme. The differences between bacterial DSB repair systems may underlie a selection for diversity when dealing with DSB. These and other features of homologous recombination in L. lactis are discussed.

Bacteriophages↗
PubMed2002

A role for RAD54B in homologous recombination in human cells.

In human somatic cells, homologous recombination is a rare event. To facilitate the targeted modification of the genome for research and gene therapy applications, efforts should be directed toward understanding the molecular mechanisms of homologous recombination in human cells. Although human genes homologous to members of the RAD52 epistasis group in yeast have been identified, no genes have been demonstrated to play a role in homologous recombination in human cells. Here, we report that RAD54B plays a critical role in targeted integration in human cells. Inactivation of RAD54B in a colon cancer cell line resulted in severe reduction of targeted integration frequency. Sensitivity to DNA-damaging agents and sister-chromatid exchange were not affected in RAD54B-deficient cells. Parts of these phenotypes were similar to those of Saccharomyces cerevisiae tid1/rdh54 mutants, suggesting that RAD54B may be a human homolog of TID1/RDH54. In yeast, TID1/RDH54 acts in the recombinational repair pathway via roles partially overlapping those of RAD54. Our findings provide the first genetic evidence that the mitotic recombination pathway is functionally conserved from yeast to humans.

Base Sequence↗
PubMed2001

Homologous recombination in extrachromosomal plasmid substrates is not suppressed by p53.

We and others reported previously that the tumor suppressor p53 down-regulates spontaneous homologous recombination in chromosomally integrating plasmid substrates, but how p53 affects homology-dependent repair of DNA double-strand breaks has not been established. Furthermore, it has been hypothesized that p53 may suppress homologous recombination by direct interaction with recombination intermediates, but it is not known whether p53 directly acts on extrachromosomal plasmid substrates. In the present study, we asked whether p53 can suppress extrachromosomal spontaneous and double-strand break-induced homologous recombination. A plasmid shuttle assay was employed utilizing episomally replicating substrates, which carried mutated tandem repeats of a CAT reporter gene. Spontaneous homologous recombination and homology-dependent repair of double-strand breaks induced by the I-SceI nuclease led to reconstitution of the reporter. Extrachromosomal homologous recombination was found to proceed independently of the p53 status of isogenic mouse fibroblast lines, contrasting the p53-mediated suppression of chromosomal recombination. The lack of p53 effect applied not only to the dominating single-strand annealing pathway, which is Rad51-independent, but also to Rad51-dependent gene conversion events. Comparison of homologous and non-homologous recombination frequencies revealed similar contributions to the repair of I-SceI-induced breaks irrespective of p53 status. Our results are consistent with a model in which the regulation of homologous recombination by p53 is restricted to the highly ordered chromosomal chromatin structure. These data may serve as a cautionary note for future investigations using solely extrachromosomal model systems to address DNA repair in intact cells.

Animals↗
PubMed2004

The Saccharomyces cerevisiae recombination enhancer biases recombination during interchromosomal mating-type switching but not in interchromosomal homologous recombination.

Haploid Saccharomyces can change mating type through HO-endonuclease cleavage of an expressor locus, MAT, followed by gene conversion using one of two repository loci, HML or HMR, as donor. The mating type of a cell dictates which repository locus is used as donor, with a cells using HML and alpha cells using HMR. This preference is established in part by RE, a locus on the left arm of chromosome III that activates the surrounding region, including HML, for recombination in a cells, an activity suppressed by alpha 2 protein in alpha cells. We have examined the ability of RE to stimulate different forms of interchromosomal recombination. We found that RE exerted an effect on interchromosomal mating-type switching and on intrachromosomal homologous recombination but not on interchromosomal homologous recombination. Also, even in the absence of RE, MAT alpha still influenced donor preference in interchromosomal mating-type switching, supporting a role of alpha 2 in donor preference independent of RE. These results suggest a model in which RE affects competition between productive and nonproductive recombination outcomes. In interchromosome gene conversion, RE enhances both productive and nonproductive pathways, whereas in intrachromosomal gene conversion and mating-type switching, RE enhances only the productive pathway.

Chromosomes, Fungal↗
PubMed1991

Thymidylate stress induces homologous recombination activity in mammalian cells.

We studied whether homologous recombination activity in mammalian cells could be induced by thymidylate stress (thymidylate deprivation). In vitro recombination activity in cell extracts was measured with pSV2neo-derived plasmids. When prior to the preparation of extracts, mouse FM3A cells were grown in 5-fluorodeoxyuridine (FdUrd), an inducer of thymidylate stress, the homologous recombination activity was significantly induced, as judged from an increase in the number of neomycin-resistant bacterial colonies. Maximum induction was observed in cells treated with 1 microM FUdR for 16 h. However, 3-8 h of treatment of FM3A cells with the drug followed by an additional 8-16-h incubation in its absence was sufficient to induce the recombination activity while slightly reducing their growth rates. These results indicate that thymidylate stress induces homologous recombination activity in mammalian cells as observed in Escherichia coli and in yeast.

Animals↗
PubMed2000

Mismatch repair is required for O(6)-methylguanine-induced homologous recombination in human fibroblasts.

O:(6)-methylguanine is responsible for homologous recombination induced by N:-methyl-N:'-nitro-N:-nitrosoguanidine (MNNG) [H. Zhang et al. (1996) CARCINOGENESIS:, 17, 2229]. To test the hypothesis that mismatch repair is causally involved in this process, we generated mismatch repair-deficient strains from a human fibroblast line containing a substrate for detecting intrachromosomal homologous recombination. The four strains selected for study exhibited greatly increased resistance to the cytotoxic effects of MNNG, which was not affected by depletion of O:(6)-alkylguanine-DNA alkyltransferase, and greatly increased sensitivity to the mutagenic effect of MNNG, suggesting that the mutagenic base modifications induced in these four cell strains by MNNG persist in their genomic DNA. Tests showed that their extracts are deficient in the repair of G:T mismatches. The frequency of homologous recombination induced by MNNG in three of these strains was significantly (5-7-fold) lower than that induced in the parental cell strain. This was not the result of a generalized defect in recombination, because when (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epox y-7,8,9, 10-tetrahydrobenzo[a]pyrene was used to induce recombination, all three lines responded with a normal or even a somewhat higher frequency than that observed in the parental strain. The lack of recombination displayed by the fourth strain was shown to result from the loss of part of the recombination substrate. The results strongly suggest that functional mismatch repair is required for MNNG-induced homologous recombination.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1994

Homologous recombination of monkey alpha-satellite repeats in an in vitro simian virus 40 replication system: possible association of recombination with DNA replication.

To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey alpha-satellite repeats. Together with the SV40-pBR322 composite vector encoding Apr and Kmr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two alpha-satellite fragments. The alpha-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity. The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E. coli galK strain DH10B after digestion with DpnI. The number of E. coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed. Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants. Similar products were also observed in the in vivo experiments with COS1 cells. The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen. Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs. These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.

Animals↗
PubMed1995

Fidelity of homologous recombination in vaccinia virus DNA.

The fidelity of homologous recombination in vaccinia virus (VV) DNA was examined by constructing a viral recombinant whose genome contained a copy of the Escherichia coli lac z gene in which the central third of the gene was repeated on either side of the VV thymidine kinase (tk) gene. In this virus, homologous DNA recombination and consequent excision of the tk gene were necessary to restore the open reading frame for beta-galactosidase and thereby to confer a lac z+ phenotype. Imprecise recombination was predicted to increase the frequency of lac z- virus. However, after several passages during which almost every viral genome underwent homologous recombination, the frequency of lac z- plaques was indistinguishable from that of a control virus that could express beta-galactosidase without prior recombination. We conclude that homologous recombination in VV DNA occurs with perfect fidelity at least 99% of the time.

DNA, Viral↗
PubMed1995

Gene replacement by homologous recombination in Mycobacterium bovis BCG.

Gene replacement by homologous recombination is a powerful tool for fundamental studies of gene function, as well as allowing specific attenuation of pathogens, but has proved difficult to achieve for Mycobacterium tuberculosis. We have used a plasmid-based test system to demonstrate the occurrence of homologous recombination in the tuberculosis vaccine strain Mycobacterium bovis BCG, and we have successfully replaced a target gene in BCG by homologous recombination, using a shuttle plasmid. Specific inactivation of selected genes will facilitate study of virulence factors and drug resistance as well as allowing rational attenuation of M. tuberculosis for the production of new vaccines.

BCG Vaccine↗
PubMed2006

Ku70 stimulates fusion of dysfunctional telomeres yet protects chromosome ends from homologous recombination.

Ku70-Ku80 heterodimers promote the non-homologous end-joining (NHEJ) of DNA breaks and, as shown here, the fusion of dysfunctional telomeres. Paradoxically, this heterodimer is also located at functional mammalian telomeres and interacts with components of shelterin, the protein complex that protects telomeres. To determine whether Ku contributes to telomere protection, we analysed Ku70(-/-) mouse cells. Telomeres of Ku70(-/-) cells had a normal DNA structure and did not activate a DNA damage signal. However, Ku70 repressed exchanges between sister telomeres - a form of homologous recombination implicated in the alternative lengthening of telomeres (ALT) pathway. Sister telomere exchanges occurred at approximately 15% of the chromosome ends when Ku70 and the telomeric protein TRF2 were absent. Combined deficiency of TRF2 and another NHEJ factor, DNA ligase IV, did not elicit this phenotype. Sister telomere exchanges were not elevated at telomeres with functional TRF2, indicating that TRF2 and Ku70 act in parallel to repress recombination. We conclude that mammalian chromosome ends are highly susceptible to homologous recombination, which can endanger cell viability if an unequal exchange generates a critically shortened telomere. Therefore, Ku- and TRF2-mediated repression of homologous recombination is an important aspect of telomere protection.

Animals↗
PubMed1999

Use of bovine satellite sequences to increase transgene integration by homologous recombination in bovine embryos.

Homologous recombination (HR) has proven to be functional in mammalian embryos. The efficiency of the HR process was tested in bovine zygotes in an attempt to increase the frequency of transgene integration using different lengths of a bovine satellite (BS) DNA flanking both ends of a neo gene marker (called BS500, BS250, and BS50) and neo alone as a control. Pronuclear microinjection at 16-19 hr post insemination (hpi) of the BS500, BS250, BS50 or neo fragments at a concentration of 1 ng/microl resulted in an increasingly negative effect on embryo development. Therefore all microinjections were performed at a single molecular concentration (320 x 10(6) molecules/ microl). After microinjection, the embryos were allowed to develop for 6 days followed by morphological and PCR analysis. The HR event was detected by PCR in 13 of the 26 embryos (43%) that developed beyond the 12-cell stage, 7/22 (31%), 9/27 (33%), and 0/25 (0%) with the BS500, BS250, BS50, and neo constructs respectively. The length of BS homology had no effect on transgene integration. However, embryos injected with BS neo constructs had significantly lower development rates than neo injected zygotes (17% more than 16 cells for BS500; 14% for BS250; 16% for BS50 compared to 32% for neo, P < 0.05, 6 replicates). These results demonstrate that BS sequences have a negative effect on embryo development and survival regardless of the amount of DNA injected. The use of HR with highly repetitive genomic sequences is therefore a feasible procedure to produce transgenic bovine embryos.

Animals↗
PubMed2001

DNA replication meets genetic exchange: chromosomal damage and its repair by homologous recombination.

Proceedings of the National Academy of Sciences Colloquium on the roles of homologous recombination in DNA replication are summarized. Current findings in experimental systems ranging from bacteriophages to mammalian cell lines substantiate the idea that homologous recombination is a system supporting DNA replication when either the template DNA is damaged or the replication machinery malfunctions. There are several lines of supporting evidence: (i) DNA replication aggravates preexisting DNA damage, which then blocks subsequent replication; (ii) replication forks abandoned by malfunctioning replisomes become prone to breakage; (iii) mutants with malfunctioning replisomes or with elevated levels of DNA damage depend on homologous recombination; and (iv) homologous recombination primes DNA replication in vivo and can restore replication fork structures in vitro. The mechanisms of recombinational repair in bacteriophage T4, Escherichia coli, and Saccharomyces cerevisiae are compared. In vitro properties of the eukaryotic recombinases suggest a bigger role for single-strand annealing in the eukaryotic recombinational repair.

Animals↗