Site-specific Recombination Process Research Articles

Ku80-deficient Cells Exhibit Excess Degradation of Extrachromosomal DNA

Mammalian cells possess a protein complex, termed DNA-PK, which binds to DNA double strand breaks in vitro. The complex consists of the heterodimeric Ku autoantigen and a DNA-dependent protein kinase, DNA-PKcs. Cell lines that are deficient for components of this complex are sensitive to ionizing radiation and have impaired V(D)J recombination, a site-specific recombination process. We have tested these cell lines for their ability to repair double strand breaks in transfected DNA. The xrs-6 cell line, which is deficient for the 80-kDa subunit of the Ku autoantigen, exhibited reduced stability of transfected DNA. Prior to obvious reductions in DNA stability, the levels of homologous recombination and DNA end joining were unaffected. However, the recovery of end joining products with precisely joined ends was reduced, with a concomitant increase in products containing deletions. Unlike the Ku80-deficient cells, no reduction in DNA stability was detected in DNA-PKcs-deficient scid cells. Scid cells also exhibited normal levels of homologous recombination and DNA end joining. These experiments implicate the Ku autoantigen, but not DNA-PKcs, in a direct role in protecting DNA ends from degradation.

The use of regular isotopy in the topological description of site-specific DNA recombination processes

A recently developed polynomial invariant of regular isotopy is used to give a topological description of site-specific DNA recombination catalyzed by Tn3 resolvase and phage Mu Gin invertase.

The fate of deleted DNA produced during programmed genomic deletion events in Tetrahymena thermophila.

Thousands of DNA deletion events occur during macronuclear development in the ciliate Tetrahymena thermophila. In two deleted genomic regions, designated M and R, the eliminated sequences form circles that can be detected by PCR. However, the circles are not normal products of the reaction pathway. The circular forms occur at very low levels in conjugating cells, but are stable. Sequencing analysis showed that many of the circles (as many as 50% of those examined) reflected a precise deletion in the M and R regions. The remaining circles were either smaller or larger and contained varying lengths of sequences derived from the chromosomal DNA surrounding the eliminated region. The chromosomal junctions left behind after deletion were more precise, although deletions in either the M or R regions can generate any of several alternative junctions (1). Some new chromosomal junctions were detected in the present study. The results suggest that the deleted segment is released as a linear DNA species that is degraded rapidly. The species is only rarely converted to the stable circles we detect. The deletion mechanism is different from those proposed for deletion events in hypotrichous ciliates (2-4), and does not reflect a conservative site-specific recombination process such as that promoted by the bacteriophage lambda integrase (5).

Site-specific integration of the temperate bacteriophage phi adh into the Lactobacillus gasseri chromosome and molecular characterization of the phage (attP) and bacterial (attB) attachment sites.

The temperate bacteriophage phi adh integrates its genome into the chromosomal DNA of Lactobacillus gasseri ADH by a site-specific recombination process. Southern hybridization analysis of BclI-digested genomic DNA from six relysogenized derivatives of the prophage-cured strain NCK102 displayed phage-chromosomal junction fragments identical to those of the lysogenic parent. The phi adh attachment site sequence, attP, was located within a 365-bp EcoRI-HindIII fragment of phage phi adh. This fragment was cloned and sequenced. DNA sequence analysis revealed striking features common to the attachment sites of other site-specific recombination systems: five direct repeats of the sequence TGTCCCTTTT(C/T) and a 14-bp inverted repeat. Oligonucleotides derived from the sequence of the attP-containing fragment enabled us to amplify predicted junction fragment sequences and thus to identify attL, attR, and attB. The core region was defined as the 16-bp sequence TACACTTCTTAGGAGG. Phage-encoded functions essential for site-specific insertion of phage phi adh were located in a 4.5-kb BclI fragment. This fragment was cloned in plasmid pSA34 to generate the insertional vector pTRK182. Plasmid pTRK182 was introduced into L. gasseri NCK102 by electroporation. Hybridization analysis showed that a single copy of pTRK182 had integrated at the attB site of the NCK102 erythromycin-resistant transformants. This is the first site-specific recombination system described in lactobacilli, as well as the first attP-based site-specific integration vector constructed for L. gasseri ADH.

Integration host factor binds specifically to multiple sites in the ompB promoter of Escherichia coli and inhibits transcription

Escherichia coli integration host factor (IHF) is a DNA-binding protein that participates in gene regulation, site-specific recombination, and other processes in E. coli and some of its bacteriophages and plasmids. In the present study, we showed that IHF is a direct negative effector of the ompB operon of E. coli. Gel retardation experiments and DNase I footprinting studies revealed that IHF binds to three sites in the ompB promoter region. In vitro transcription from ompB promoter fragments was specifically blocked by IHF. In vivo experiments showed that IHF is a negative effector of ompB expression in growing cells. Analysis of IHF binding site mutations strongly suggested that IHF binding in the ompB promoter region is necessary for the negative effects seen in vivo.

The FIS protein binds and bends the origin of chromosomal DNA replication,oriC, ofEscherichia coli

The FIS protein (factor for inversion stimulation) is known to stimulate site-specific recombination processes, such as the inversion of the G segment of bacteriophage Mu, by binding to specific enhancer sequences. It has also been shown to activate transcription from rRNA promoters both in vitro and in vivo. We have identified a specific binding site for FIS in the center of the origin of chromosomal DNA replication, oriC. The DNA bends upon FIS binding. Occupation of the FIS site and binding of DnaA, the initiator protein, to its adjacent binding site (R3) are mutually exclusive. A fis mutant strain can not be efficiently transformed with plasmids which carry and replicate from oriC, suggesting that FIS is required for minichromosome replication.

Site-specific recombination by Tn3 resolvase

Site-specific recombination processes in microbes bring about precise DNA rearrangements which have diverse and important biological functions. The sites and recombinase enzymes used for these processes fall into two distinct families. Here we described how experiments with one family, exemplified by the resolution system of transposon Tn3, have provided insight into the ways in which DNA and protein interact to bring together distant recombination sites and promote strand exchange.

Site-specific and illegitimate recombination in the oriV1 region of the F factor : DNA sequences involved in recombination and resolution

We have defined some of the sequences involved in high frequency recA-independent recombination at the oriV1 region of the F factor. Using a mobilization assay, we determined that plasmid pMB080, a pBR322 derivative bearing the PvuII- BamHI (F factor co-ordinates 45.43 to 46.0) fragment from the oriV1 region of F, contained all sequences necessary to undergo efficient site-specific recombination with the F derivative pOX38, which retains the oriV1 region. We constructed a series of pMB080 deletions in vitro using exonucleases S 1 and Bal31. Deletions removing a ten base-pair sequence, which forms part of an inverted repeat segment located 62 base-pairs to the left of the NcoI site (45.87) within the cloned fragment, totally eliminated the recA-independent recombination reaction. Other deletions differentially affected both the frequency and stability of cointegrate molecules formed by the site-specific recombination system. The F factor oriV1 region is involved also in low-frequency recombination with several sites on pBR322 and related plasmids. We have determined the precise location of these recombination sites within oriV1 by DNA sequencing. These studies revealed that recombination always took place within an eight base-pair spacer region between the ten base-pair inverted repeats found to be important for oriV1- oriV1 interactions. We propose that the low-efficiency recombination between pBR322 and pOX38 results from the ability of the F site-specific recombination apparatus to weakly recognize and interact with sequences that bear some resemblance to the normal oriV1 recognition elements. Furthermore, we suggest, by analogy with the lambda paradigm, that the nucleotide sequences at the junctions of secondary site recombinants define at least one crossover site used during the normal site-specific recombination process.