Orientation Dependence in Homologous Recombination

Homologous recombination was investigated in Escherichia coli with two plasmids, each carrying the homologous region (two defective neo genes, one with an amino-end deletion and the other with a carboxyl-end deletion) in either direct or inverted orientation. Recombination efficiency was measured in recBC sbcBC and recBC sbcA strains in three ways. First, we measured the frequency of cells carrying neo(+) recombinant plasmids in stationary phase. Recombination between direct repeats was much more frequent than between inverted repeats in the recBC sbcBC strain but was equally frequent in the two substrates in the recBC sbcA strain. Second, the fluctuation test was used to exclude bias by a rate difference between the recombinant and parental plasmids and led to the same conclusion. Third, direct selection for recombinants just after transformation with or without substrate double-strand breaks yielded essentially the same results. Double-strand breaks elevated recombination in both the strains and in both substrates. These results are consistant with our previous findings that the major route of recombination in recBC sbcBC strains generates only one recombinant DNA from two DNAs and in recBC sbcA strains generates two recombinant DNAs from two DNAs.     

Plasmid Recombination in a Rad52 Mutant of Saccharomyces Cerevisiae

Using plasmids capable of undergoing intramolecular recombination, we have compared the rates and the molecular outcomes of recombination events in a wild-type and a rad52 strain of Saccharomyces cerevisiae. The plasmids contain his3 heteroalleles oriented in either an inverted or a direct repeat. Inverted repeat plasmids recombine approximately 20-fold less frequently in the mutant than in the wild-type strain. Most events from both cell types have continuous coconversion tracts extending along one of the homologous segments. Reciprocal exchange occurs in fewer than 30% of events. Direct repeat plasmids recombine at rates comparable to those of inverted repeat plasmids in wild-type cells. Direct repeat conversion tracts are similar to inverted repeat conversion tracts in their continuity and length. Inverted and direct repeat plasmid recombination differ in two respects. First, rad52 does not affect the rate of direct repeat recombination as drastically as the rate of inverted repeat recombination. Second, direct repeat plasmids undergo crossing over more frequently than inverted repeat plasmids. In addition, crossovers constitute a larger fraction of mutant than wild-type direct repeat events. Many crossover events from both cell types are unusual in that the crossover HIS3 allele is within a plasmid containing the parental his3 heteroalleles.     

The Effects of Insertions on Mammalian Intrachromosomal Recombination

We examined the effects of insertion mutations on intrachromosomal recombination. A series of mouse L cell lines carrying mutant herpes simplex virus thymidine kinase (tk) heteroalleles was generated; these lines differed in the nature of their insertion mutations. In direct repeat lines with different large insertions in each gene, there was a 20-fold drop in gene conversion rate and only a five-fold drop in crossover rate relative to the analogous rates in lines with small insertions in each gene. Surprisingly, in direct repeat lines carrying the same large insertion in each gene, there was a larger drop in both types of recombination. When intrachromosomal recombination between inverted repeat tk genes with different large insertions was examined, we found that the rate of gene conversion dropped five-fold relative to small insertions, while the rate of crossing over was unaffected. The differential effects on conversion and crossing over imply that gene conversion is more sensitive to insertion mutation size. Finally, the fraction of gene conversions associated with a crossover increased from 2% for inverted repeats with small insertions to 18% for inverted repeats with large insertions. One interpretation of this finding is that during intrachromosomal recombination in mouse cells long conversion tracts are more often associated with crossing over.     

Somatic and Meiotic Chromosomal Recombination between Inverted Duplications in Transgenic Tobacco Plants

Homologous recombination has been extensively studied in bacteria, yeast, and more recently in animal cells, but little is known about this process in plants. We present here an analysis of meiotic and somatic chromosomal recombination between closely linked inverted duplications located on a single chromosomal region in tobacco. Transgenic tobacco lines were constructed by Agrobacterium transformation with plasmid vectors containing a functional hygromycin phosphotransferase (hyg) selectable marker flanked by a pair of defective neomycin phosphotransferase (neo) genes positioned as inverted repeats. As each neo gene is mutated in a different site, recombination between the two defective genes can be detected following selection for kanamycin-resistant plant cells. The recombination substrates were designed to allow investigation into the nature of molecular events underlying homologous recombination by restriction endonuclease analysis. Chromosomal recombination was studied in mitotically dividing cells (cultured leaf mesophyll cells) and after meiosis (germinated seedlings). Spontaneous somatic recombinants were recovered at frequencies between ~3 x 10-5 to 10-6 events per cell. Low dose [gamma] irradiation of somatic cells resulted in a threefold maximum increase in the recovery of recombinants. Recombinants were also detected at low frequency when transgenic T3 seeds were germinated under kanamycin selection. DNA gel blot analyses demonstrated that homologous recombination occurred mainly as gene conversion unassociated with reciprocal exchange, although a variety of other events including gene coconversion were also observed.     

Gene conversion in the Escherichia coli RecF pathway: a successive half crossing-over model.

Summary Gene conversion - apparently non-reciprocal transfer of sequence information between homologous DNA sequences - has been reported in various organisms. Frequent association of gene conversion with reciprocal exchange (crossing-over) of the flanking sequences in meiosis has formed the basis of the current view that gene conversion reflects events at the site of interaction during homologous recombination. In order to analyze mechanisms of gene conversion and homologous recombination in an Escherichia coli strain with an active RecF pathway (recBC sbcBC), we first established in cells of this strain a plasmid carrying two mutant neo genes, each deleted for a different gene segment, in inverted orientation. We then selected kanamycin-resistant plasmids that had reconstituted an intact neo ⁺ gene by homologous recombination. We found that all the neo ⁺ plasmids from these clones belonged to the gene-conversion type in the sense that they carried one neo ⁺ gene and retained one of the mutant neo genes. This apparent gene conversion was, however, only very rarely accompanied by apparent crossing-over of the flanking sequences. This is in contrast to the case in a rec ⁺ strain. or in a strain with an active RecE pathway (recBC sbcA). Our further analyses, especially comparisons with apparent gene conversion in the rec ⁺ strain, led us to propose a mechanism for this biased gene conversion. This successive half crossing-over model proposes that the elementary recombinational process is half crossing;-over in the sense that it generates only one recombinant DNA duplex molecule, and leaves one or two free end(s), out of two parental DNA duplexes. The resulting free end is, the model assumes, recombinogenic and frequently engages in a second round of half crossing-over with the recombinant duplex. The products resulting from such interaction involving two molecules of the plasmid would be classified as belonging to the gene-conversion type without crossing-over. We constructed a dimeric molecule that mimics the intermediate form hypothesized in this model and introduced it into cells. Biased gene conversion products were obtained in this reconstruction experiment. The half crossing-over mechanism can also explain formation of huge linear multimers of bacterial plasmids, the nature of transcribable recombination products in bacterial conjugation, chromosomal gene conversion not accompanied by flanking exchange (like that in yeast mating-type switching), and antigenic variation in microorganisms.     

Homologous Recombination between Autonomously Replicating Plasmids in Mammalian Cells

The ability of autonomously replicating plasmids to recombine in mammalian cells was investigated. Two deletion plasmids of the eukaryotic-prokaryotic shuttle vector pSV2neo were cotransfected into transformed monkey COS cells. Examination of the low molecular weight DNA isolated after 48 hr of incubation revealed that recombination between the plasmids had occurred. The DNA was also used to transform recA- E. coli. Yield of neoR colonies signified homologous recombination. Examination of the plasmid DNA from these colonies confirmed this view. Double-strand breaks in one or both of the input plasmids at the sites of deletion resulted in an enhancement of recombination frequency. The recombination process yielded monomeric and dimeric molecules. Examination of these molecules revealed that reciprocal recombination as well as gene conversion events were involved in the generation of plasmids bearing an intact neo gene. The COS cell system we describe is analogous to study of bacteriophage recombination and yeast random-spore analysis.     

Use of a dominant rpsL allele conferring streptomycin dependence for positive and negative selection in Thermus thermophilus

A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.     

Use of a Dominant rpsL Allele Conferring Streptomycin Dependence for Positive and Negative Selection in Thermus thermophilus

A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.     

In vivo linearization and autonomous replication of plasmids containing human telomeric DNA in Aspergillus nidulans

Plasmids containing two inverted 0.6-kb stretches of human telomeric repeats transform Aspergillus nidulans at frequencies characteristic of autonomously replicating vectors. Transformation frequency is not affected when the plasmids are linearized in vitro prior to transformation by cutting between the inverted repeats. Southern analysis reveals the presence of a homogeneous pool of linear plasmid molecules in mycelium of transformants. Addition of the AMA1 plasmid replicator to the telomere-containing plasmids has only a minor effect on transformation. The phenotypic stability of the transformants is low. However, unlike conventional replicative transformants containing AMA1-bearing plasmids, these transformants are prone to spontaneous stabilization which occurs predominantly by conversion of the mutant chromosomal allele of the marker gene to the plasmid-borne allele. The data strongly suggest that telomeric DNA can act as a plasmid replicator. An alternative interpretation is that autonomous replication of linear DNA fragments, in contrast to covalently closed supercoiled molecules, does not require any special replicator sequences.     

Intra-chromosomal gene conversion induced by a DNA double-strand break in Saccharomyces cerevisiae

We have stimulated mitotic and meiotic gene conversion between non-tandem direct repeats of ADE4 by a defined double-strand break imparted in vivo to one of two copies of the gene. The experimental design permitted us to distinguish unambiguously between reciprocal intra-chromosomal crossing over and non-reciprocal break-join events that could accompany the induced conversions. We observed that (1) less than 10% of the induced conversion events are accompanied by intra-chromosomal crossing over in both mitosis and meiosis; (2) non-reciprocal break-join is not stimulated by the double-strand breaks; (3) a double-strand break in meiosis is repaired off intra-chromosomal homology (if available) with approximately sevenfold preference over repair off the homologous chromosome. Our observations, analyzed in the light of previous investigations of spontaneous inter and intra-chromosomal crossing over and gene conversion, lead to the view that chromosomal configuration constrains intra-chromosomal crossing over accompanying conversion between closely spaced repeated genes during resolution of the conversion intermediate.     

In vivo linearization and autonomous replication of plasmids containing human telomeric DNA in Aspergillus nidulans

Plasmids containing two inverted 0.6-kb stretches of human telomeric repeats transform Aspergillus nidulans at frequencies characteristic of autonomously replicating vectors. Transformation frequency is not affected when the plasmids are linearized in vitro prior to transformation by cutting between the inverted repeats. Southern analysis reveals the presence of a homogeneous pool of linear plasmid molecules in mycelium of transformants. Addition of the AMA1 plasmid replicator to the telomere-containing plasmids has only a minor effect on transformation. The phenotypic stability of the transformants is low. However, unlike conventional replicative transformants containing AMA1-bearing plasmids, these transformants are prone to spontaneous stabilization which occurs predominantly by conversion of the mutant chromosomal allele of the marker gene to the plasmid-borne allele. The data strongly suggest that telomeric DNA can act as a plasmid replicator. An alternative interpretation is that autonomous replication of linear DNA fragments, in contrast to covalently closed supercoiled molecules, does not require any special replicator sequences. Received: 13 January 1998 / Accepted: 10 June 1998     

Stability of an inverted repeat in a human fibrosarcoma cell.

Deletions and rearrangements of DNA sequences within the genome of human cells result in mutations associated with human disease. We have developed a selection system involving a neo gene containing a DNA sequence inserted into the NcoI site that can be used to quantitatively assay deletion of this sequence from the chromosome. The spontaneous deletion from the neo gene of a 122 bp inverted repeat occurred at a rate of 2.1 x 10(-8) to <3.1 x 10(-9) revertants/cell/generation in three different cell lines. Deletion of the 122 bp inverted repeat occurred between 6 bp flanking direct repeats. Spontaneous deletion of a 122 bp non-palindromic DNA sequence flanked by direct repeats was not observed, indicating a rate of deletion of <3.1 x 10(-9) revertants/cell/generation. This result demonstrates that a 122 bp inverted repeat can exhibit a low level of instability in some locations in the chromosome of a human cell line.     

Gene conversion associated with site-specific recombination in yeast plasmid pSR1.

A circular DNA plasmid, pSR1, isolated from Zygosaccharomyces rouxii has a pair of inverted repeats consisting of completely homologous 959-base pair (bp) sequences. Intramolecular recombination occurs frequently at the inverted repeats in cells of Saccharomyces cerevisiae, as well as in Z. rouxii, and is catalyzed by a protein encoded by the R gene of its own genome. The recombination is, however, independent of the RAD52 gene of the host genome. A site for initiation of the intramolecular recombination in the S. cerevisiae host was delimited into, at most, a 58-bp region in the inverted repeats by using mutant plasmids created by linker insertion. The 58-bp region contains a pair with 14-bp dyad symmetry separated by a 3-bp spacer sequence. The recombination initiated at this site was accompanied by a high frequency of gene conversion (3 to 50% of the plasmid clones examined). Heterogeneity created by the linker insertion or by a deletion (at most 153 bp so far tested) at any place on the inverted repeats was converted to a homologous combination by the gene conversion, even in the rad52-1 mutant host. A mechanism implying branch migration coupled with DNA replication is discussed.     

Genetic and Physical Analysis of Double-Strand Break Repair and Recombination in Saccharomyces Cerevisiae

We have investigated HO endonuclease-induced double-strand break (DSB) recombination and repair in a LACZ duplication plasmid in yeast. A 117-bp MATa fragment, embedded in one copy of LACZ, served as a site for initiation of a DSB when HO endonuclease was expressed. The DSB could be repaired using wild-type sequences located on a second, promoterless, copy of LACZ on the same plasmid. In contrast to normal mating-type switching, crossing-over associated with gene conversion occurred at least 50% of the time. The proportion of conversion events accompanied by exchange was greater when the two copies of LACZ were in direct orientation (80%), than when inverted (50%). In addition, the fraction of plasmids lost was significantly greater in the inverted orientation. The kinetics of appearance of intermediates and final products were also monitored. The repair of the DSB is slow, requiring at least an hour from the detection of the HO-cut fragments to completion of repair. Surprisingly, the appearance of the two reciprocal products of crossing over did not occur with the same kinetics. For example, when the two LACZ sequences were in the direct orientation, the HO-induced formation of a large circular deletion product was not accompanied by the appearance of a small circular reciprocal product. We suggest that these differences may reflect two kinetically separable processes, one involving only one cut end and the other resulting from the concerted participation of both ends of the DSB.     

Intrachromosomal Recombination in Saccharomyces cerevisiae : Reciprocal Exchange in an Inverted Repeat and Associated Gene Conversion

Intrachromosomal gene conversion has not shown a strong association with reciprocal exchanges. However, reciprocal exchanges do occur between intrachromosomal repeats. To understand the relationship between reciprocal exchange and gene conversion in repeated sequences the recombination behavior of an inverted repeat was studied. We have found that in one orientation a single copy of the kanr gene of the bacterial transposon Tn903 flanked by part of the inverted repeats IS903 does not give G418 resistance in Saccharomyces cerevisiae. A reciprocal exchange in the IS903 repeats inverts the kanr gene, which then gives G418 resistance in a single copy. Using this as a selection for intrachromosomal reciprocal exchange we have introduced multiple restriction site heterologies into the IS903 repeats and examined the crossover products for associated gene conversions. Approximately 50% of crossovers, both in mitosis and meiosis, were associated with a gene conversion. This suggests that these crossovers result from an intermediate that gives a gene conversion in 50% of the events, that is, both reciprocal exchange and gene conversion between repeated sequences have a common origin. The data are most consistent with a heteroduplex mismatch repair mechanism.     

Homologous and nonhomologous recombination resulting in deletion: effects of p53 status, microhomology, and repetitive DNA length and orientation

Repetitive DNA elements frequently are precursors to chromosomal deletions in prokaryotes and lower eukaryotes. However, little is known about the relationship between repeated sequences and deletion formation in mammalian cells. We have created a novel integrated plasmid-based recombination assay to investigate repeated sequence instability in human cells. In a control cell line, the presence of direct or inverted repeats did not appreciably influence the very low deletion frequencies (2 x 10(-7) to 9 x 10(-7)) in the region containing the repeat. Similar to what has been observed in lower eukaryotes, the majority of deletions resulted from the loss of the largest direct repeat present in the system along with the intervening sequence. Interestingly, in closely related cell lines that possess a mutant p53 gene, deletion frequencies in the control and direct-repeat plasmids were 40 to 300 times higher than in their wild-type counterparts. However, mutant p53 cells did not preferentially utilize the largest available homology in the formation of the deletion. Surprisingly, inverted repeats were approximately 10,000 times more unstable in all mutant p53 cells than in wild-type cells. Finally, several deletion junctions were marked by the addition of novel bases that were homologous to one of the preexisting DNA ends. Contrary to our expectations, only 6% of deletions in all cell lines could be classified as arising from nonhomologous recombination.     

Improvements to gene deletion in the fungal pathogen Cryptococcus neoformans: absence of Ku proteins increases homologous recombination, and co-transformation of independent DNA molecules allows rapid complementation of deletion phenotypes

Cryptococcus neoformans is a pathogenic fungus that is relatively amenable to molecular genetic analysis, including gene deletion. However, rates of homologous recombination can be low, so obtaining specific gene deletion transformants is challenging. We have utilized two new technologies, cku deletion strains to improve the efficiency of gene deletions in this organism, and co-transformations. The Ku70-Ku80 heterodimer is predicted to be an essential part of the non-homologous end-joining process in C. neoformans. Here we show that a deletion in one or both of these proteins results in an increase in the rates of homologous recombination. Importantly, we demonstrate that after generation of a strain with a particular deletion of interest, the cku deletion can be removed by mating and segregation. We also utilize co-transformation of wild-type genes and selectable markers on separate linear DNA molecules to complement a deletion event. We show that co-transformation results in the successful restoration of wild-type phenotype, though variations in this phenotype often occur.     

Different pathways of homologous recombination are used for the repair of double-strand breaks within tandemly arranged sequences in the plant genome

Different DNA repair pathways that use homologous sequences in close proximity to genomic double-strand breaks (DSBs) result in either an internal deletion or a gene conversion. We determined the efficiency of these pathways in somatic plant cells of transgenic Arabidopsis lines by monitoring the restoration of the beta-glucuronidase (GUS) marker gene. The transgenes contain a recognition site for the restriction endonuclease I-SceI either between direct GUS repeats to detect deletion formation (DGU.US), or within the GUS gene to detect gene conversion using a nearby donor sequence in direct or inverted orientation (DU.GUS and IU.GUS). Without expression of I-SceI, the frequency of homologous recombination (HR) was low and similar for all three constructs. By crossing the different lines with an I-SceI expressing line, DSB repair was induced, and resulted in one to two orders of magnitude higher recombination frequency. The frequencies obtained with the DGU.US construct were about five times higher than those obtained with DU.GUS and IU.GUS, irrespective of the orientation of the donor sequence. Our results indicate that recombination associated with deletions is the most efficient pathway of homologous DSB repair in plants. However, DSB-induced gene conversion seems to be frequent enough to play a significant role in the evolution of tandemly arranged gene families like resistance genes.