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PubMed1984

An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.

To identify specific cellular factors which could be required during the synthesis of retroviral DNA, we have studied the replication of murine leukemia virus in mouse cells temperature sensitive for cell DNA synthesis (M. L. Slater and H. L. Ozer, Cell 7:289-295, 1976) and in several of their revertants. This mutation has previously been mapped on the X chromosome. We found that a short incubation of mutant cells at a nonpermissive temperature (39 degrees C) during the early part of the virus cycle (between 0- to 20-h postinfection) greatly inhibited virus production. This effect was not observed in revertant or wild-type cells. Molecular studies by the Southern transfer procedure of the unintegrated viral DNA synthesized in these cells at a permissive (33 degrees C) or nonpermissive temperature revealed that the levels of linear double-stranded viral DNA (8.8 kilobase pairs) were nearly identical in mutant or revertant cells incubated at 33 or 39 degrees C. However, the levels of two species of supercoiled viral DNA (with one or two long terminal repeats) were significantly lower in mutant cells incubated at 39 degrees C than in mutant cells incubated at 33 degrees C or in revertant cells incubated at 39 degrees C. Pulse-chase experiments showed that linear viral DNA made at 39 degrees C could not be converted into supercoiled viral DNA in mutant cells after a shift down to 33 degrees C. In contrast, such conversion was observed in revertant cells. Restriction endonuclease analysis did not detect differences in the structure of linear viral DNA made at 39 degrees C in mutant cells as compared to linear viral DNA isolated from the same cells at 33 degrees C. However, linear viral DNA made at 39 degrees C in mutant cells was poorly infectious in transfection assays. Taken together, these results strongly suggest that this X-linked gene, affecting mouse cell DNA synthesis, is operating in the early phase of murine leukemia virus replication. It seems to affect the level of production of unintegrated linear viral DNA only slightly while greatly reducing the infectivity of these molecules. In contrast, the accumulation of supercoiled viral DNA and subsequent progeny virus production are greatly reduced. Our pulse-chase experiments suggest that the apparent, but not yet identified, defect in linear viral DNA molecules might be responsible for their subsequent impaired circularization.

Animals↗

PubMed1990

Direction of the DNA-unwinding reaction catalysed by Escherichia coli DNA helicase II.

The direction of the DNA-unwinding reaction catalysed by Escherichia coli DNA helicase II was studied using gapped linear DNA molecules with short duplex ends as substrate. The results suggest that DNA helicase II unwinds with 3'-5' polarity relative to the single strand of the DNA partial duplex. At high enzyme DNA ratio the enzyme also unwinds the duplex connected to the 3' end of the single strand and, as further studies show, fully duplex linear DNA. The fraction of DNA unwound decreases as the length of the duplex substrate increases. The preference of DNA helicase II for a short duplex can obscure the fact that the typical substrate is duplex connected to the 5' end of a single strand.

Adenosine Triphosphatases↗

PubMed2006

Sequence identity of the direct repeats, DR1 and DR2, contributes to the discrimination between primer translocation and in situ priming during replication of the duck hepatitis B virus.

There are two mutually exclusive pathways for plus-strand DNA synthesis in hepadnavirus reverse transcription. The predominant pathway gives rise to relaxed circular DNA, while the other pathway yields duplex linear DNA. At the completion of minus-strand DNA synthesis, the final RNase H cleavage generates the plus-strand primer at direct repeat 1 (DR1). A small fraction of viruses make duplex linear DNA after initiating plus-strand DNA synthesis from this site, a process called in situ priming. To make relaxed circular DNA, a template switch is necessary for the RNA primer generated at DR1 to initiate plus-strand DNA synthesis from the direct repeat 2 (DR2) located near the opposite end of the minus-strand DNA, a process called primer translocation. We are interested in understanding the mechanism that discriminates between these two processes. Previously, we showed that a small DNA hairpin forms at DR1 in the avihepadnaviruses and acts as an inhibitor of in situ priming. Here, using genetic approaches, we show that sequence identity between DR1 and DR2 is necessary, but not sufficient for primer translocation in the duck hepatitis B virus. The discrimination between in situ priming and primer translocation depends upon suppression of in situ priming, a process that is dependent upon both sequence identity between DR1 and DR2, and the presence of the hairpin at DR1. Finally, our analysis indicates the entire RNA primer can contribute to primer translocation and is translocated to DR2 before initiation of plus-strand DNA synthesis from that site.

Animals↗

PubMed1984

Formation of inverted dimer plasmids after transformation of yeast with linearized plasmid DNA.

The formation of an inverted dimer plasmid on transformation with linear molecules is formally analogous to the fusion of the daughters of a broken chromosome at their broken ends. In the latter case, this leads to the formation of a dicentric chromosome, which could break at anaphase. Hence the process is cyclic. Similarly, when our linear molecules are modified by the addition of a cloned yeast centromere, dicentric inverted dimers are not obtained. Instead, we obtain monocentric plasmids with partial duplication and deletion that apparently derive from a process of fusion, bridge-breakage, and fusion. This is not surprising, since it is known that dicentric plasmids undergo breakage in yeast (Mann and Davis 1983). However, any apparent similarity of this process to that which occurs with a broken chromosome in maize must be tempered by the special nature of the transformation process. Most significantly, inverted dimers are rare when sonicated carrier DNA is not present during the transformation. This requirement is not understood, but it is a condition that may not be met in a yeast cell harboring a broken chromosome. It is possible that carrier DNA induces a repair process that results in fusion. On the other hand, a property of the transformation process that results in an inhibition of fusion may be overcome by the presence of carrier DNA. Most inverted dimers are apparently formed from an interaction between two input linear molecules. We cannot rule out the possibility that a minor fraction derive from a single molecule. Thus, the fusion of two input molecules is a much more efficient process than a replicative process that could occur with single linear molecule. For a similar fusion process to occur with a broken yeast chromosome, replication would be required. We do not know if a broken yeast chromosome can replicate. Evidence consistent with the presence of a breakage-fusion-bridge process in yeast has been obtained through the formation of dicentric chromosomes via meiotic recombination (Haber et al. 1984). Spores from these meioses sometimes give rise to a clone that is mixed for markers of the chromosome that could have been dicentric. A process of fusion-bridge-breakage could account for the formation of some of these mixed clones. However, the dicentric chromosomes apparently often survive meiotic disjunction and break in the spore's first mitotic anaphase or possibly in a later generation. Thus, the interpretation of the origin of these mixed clones is uncertain. Some aspects of the fusion process are especially intriguing.(ABSTRACT TRUNCATED AT 400 WORDS)

Chromosome Mapping↗

PubMed1996

Mapping of the ribosomal operons on the linear chromosomal DNA of Streptomyces ambofaciens DSM40697.

Streptomyces ambofaciens DSM40697 possess six rRNA gene clusters which can be distinguished by probing BfrI-digested total DNA with the PCR-amplified l6S-23S ribosomal genet internal transcribed spacer. The six rrn loci of S. ambofaciens were cloned as recombinant cosmids and located on the AseI-Dŕal physical map of the linear chromosomal DNA. For five of the six ribosomal gene sets, the transcriptional orientation was determined relative to the physical map and was shown to be divergent away from an oriC-like locus.

Base Sequence↗

PubMed1981

Nucleotide sequence of cloned unintegrated avian sarcoma virus DNA: viral DNA contains direct and inverted repeats similar to those in transposable elements.

We have determined the nucleotide sequence of portions of two circular avian sarcoma virus (ASV) DNA molecules cloned in a prokaryotic host--vector system. The region whose sequence was determined represents the circle junction site--i.e., the site at which the ends of the unintegrated linear DNA are fused to form circular DNA. The sequence from one cloned molecule, SRA-2, shows that the circle junction site is the center of a 330-base-pair (bp) tandem direct repeat, presumably representing the fusion of the long terminal repeat (LTR) units known to be present at the ends of the linear DNA. The circle junction site is also the center of a 15-bp imperfect inverted repeat, which thus appears at the boundaries of the LTR. The structure of ASV DNA--unique coding region flanked by a direct repeat that is, in turn, terminated with a short inverted repeat--is very similar to the structure of certain transposable elements. Several features of the sequence imply that circularization to form the SRA-2 molecule occurred without loss of information from the linear DNA precursor. Circularization of another cloned viral DNA molecule, SRA-1, probably occurred by a different mechanism. The circle junction site of the SRA-1 molecule has a 63-bp deletion, which may have arisen by a mechanism that is analogous to the integration of viral DNA into the host genome. Flanking one side of the tandem direct repeat is the binding site for tRNATrp, the previously described primer for synthesis of the first strand of viral DNA. The other side of the direct repeat is flanked by a polypurine tract, A-G-G-G-A-G-G-G-G-G-A, which may represent the position of the primer for synthesis of the second strand of viral DNA. An A+T-rich region, upstream from the RNA capping site, and the sequence A-A-T-A-A-A are present within the direct repeat sequence. These sequences may serve as a promoter site and poly(A) addition signal, respectively, as proposed for other eukaryotic transcription units.

Avian Sarcoma Viruses↗

PubMed1984

Detection of circular and linear herpesvirus DNA molecules in mammalian cells by gel electrophoresis.

A simple gel technique is described for the detection of large, covalently closed, circular DNA molecules in eucaryotic cells. The procedure is based on the electrophoretic technique of Eckhardt (T. Eckhardt, Plasmid 1:584-588, 1978) for detecting bacterial plasmids and has been modified for the detection of circular and linear extrachromosomal herpesvirus genomes in mammalian cells. Gentle lysis of suspended cells in the well of an agarose gel followed by high-voltage electrophoresis allows separation of extrachromosomal DNA from the bulk of cellular DNA. Circular viral DNA from cells which carry the genomes of Epstein-Barr virus, Herpesvirus saimiri, and Herpesvirus ateles can be detected in these gels as sharp bands which comigrate with bacterial plasmid DNA of 208 kilobases. Epstein-Barr virus producer cell lines also show a sharp band of linear 160-kilobase DNA. The kinetics of the appearance of this linear band after induction of viral replication after temperature shift parallels the known kinetics of Epstein-Barr virus production in these cell lines. Hybridization of DNA after transfer to filters shows that the circular and linear DNA bands are virus specific and that as little as 0.25 Epstein-Barr virus genome per cell can be detected. The technique is simple, rapid, and sensitive and requires relatively low amounts of cells (0.5 X 10(6) to 2.5 X 10(6)).

Animals↗

PubMed1997

Gene targeting by linear duplex DNA frequently occurs by assimilation of a single strand that is subject to preferential mismatch correction.

To study targeted recombination, a single linear 2-kb fragment of LEU2 DNA was liberated from a chromosomal site within the nucleus of Saccharomyces cerevisiae, by expression of the site-specific HO endonuclease. Gene targeting was scored by gene conversion of a chromosomal leu2 mutant allele by the liberated LEU2 fragment. This occurred at a frequency of only 2 x 10(-4), despite the fact that nearly all cells successfully repaired, by single-strand annealing, the chromosome break created by liberating the fragment. The frequency of Leu+ recombinants was 6- to 25-fold higher in pms1 strains lacking mismatch repair. In 70% of these cases, the colony was sectored for Leu+/Leu-. Similar results were obtained when a 4. 1-kb fragment containing adjacent LEU2 and ADE1 genes was liberated, to convert adjacent leu2 and ade1 mutations on the chromosome. These results suggest that a linear fragment is not assimilated into the recipient chromosome by two crossovers each close to the end of the fragment; rather, heteroduplex DNA between the fragment and the chromosome is apparently formed over the entire region, by the assimilation of one of the two strands of the linear duplex DNA. Moreover, the recovery of Leu+ transformants is frequently defeated by the cell's mismatch repair machinery; more than 85% of mismatches in heteroduplex DNA are corrected in favor of the resident, unbroken (mutant) strand.

3-Isopropylmalate Dehydrogenase↗

PubMed1986

Characterization of a defective phage system for the analysis of bacteriophage T4 DNA replication origins.

We have developed a defective phage system for the isolation and analysis of phage T4 replication origins based on the T4-mediated transduction of plasmid pBR322. During the initial infection of a plasmid-containing cell, recombinant plasmids with T4 DNA inserts are converted into fully modified linear DNA concatamers that are packaged into T4 phage particles, to create defective phage (transducing particles). In order to select T4 replication origins from genomic libraries of T4 sequences cloned into the plasmid pBR322, we searched for recombinant plasmids that transduce with an unusually high efficiency, reasoning that this should select for T4 sequences that function as origins on plasmid DNA after phage infection. We also selected for defective phage that can propagate efficiently with the aid of a coinfecting helper phage during subsequent rounds of phage infection, which should select for T4 sequences that can function as origins on the linear DNA present in the defective phage. Several T4 inserts were isolated repeatedly in one or both of these selective procedures, and these were mapped to particular locations on the T4 genome. When plasmids were selected in this way from genomic libraries constructed using different restriction nucleases, they contained overlapping segments of the T4 genome, indicating that the same T4 sequences were selected. The inserts in two of the selected plasmids permit a very high frequency of transduction from circular plasmids; these have been shown to contain a special type of T4 replication origin.

DNA Replication↗

PubMed1995

A 7-base-pair sequence protects DNA from exonucleolytic degradation in Lactococcus lactis.

Linear DNA molecules are subject to degradation by various exonucleases in vivo unless their ends are protected. It has been demonstrated that a specific 8-bp sequence, 5'-GCTGGTGG-3', named Chi, can protect linear double-stranded DNA from the major Escherichia coli exonuclease RecBCD. Chi protects linear replication products of rolling-circle plasmids from RecBCD degradation in vivo, in agreement with observations in vitro. A unique 7-bp sequence, 5'-GCGCGTG-3', is shown to protect similar replication products from degradation in Lactococcus lactis strains but not in more distantly related Gram-positive bacteria. The properties of this sequence in L. lactis correspond to those of a Chi site. Linear plasmid replication products have been detected in numerous prokaryotes, suggesting the widespread existence of short species-specific sequences that preserve linear DNA from extensive degradation by host cell exonucleases.

Base Composition↗

PubMed1994

Genes of the linear mitochondrial DNA of Williopsis mrakii: coding sequences for a maturase-like protein, a ribosomal protein VAR1 homologue, cytochrome oxidase subunit 2 and methionyl tRNA.

The mitochondrial DNA (mtDNA) in some yeasts has a linear structure with inverted terminal repeats closed by a single-stranded loop. These mtDNAs have generally a constant gene order, beginning with a small ribosomal RNA gene at the right end and terminating with a cytochrome oxidase subunit 2 gene (COX2) at the left end, independently of the wide variation in genome size. In the mtDNAs from several species of the genus Williopsis, we found an additional open reading frame, ORF1, which was homologous to the Saccharomyces cerevisiae RF1 gene encoding a group I intron maturase-like protein. ORF1 genes from W. mrakii and W. suaveolens were mapped and sequenced. Next to ORF1, COX2 and methionyl tRNA genes were present on the opposite strand. The same relative positions of genes in the mtDNAs so far examined suggests that the constancy of gene order is generally conserved also at the level of individual tRNA genes. We identified another open reading frame, ORF2, in W. mrakii mtDNA. It was mapped next to the cytochrome oxidase subunit 3 gene. Rich in adenine-thymine bases, ORF2 appears to be a homologue of the VAR1 gene which codes for a small ribosomal subunit protein in S. cerevisiae mitochondria. Nucleotide sequences data have been deposited in the EmBL data library under the following Accession Numbers: X66594 (Apocytochrome b and ORF2 genes of W. mrakii), X66595 (ORF1, tRNA-Met and COX2 genes of W. mrakii), X73415 (tRNA-Met and COX2 genes of W. suaveolens), X73416 (ORF1 gene of W. suaveolens) and X73414 (tRNA-Met and COX2 genes of P. jadinii).

Amino Acid Sequence↗

PubMed1985

Interactions of benzo(a)pyrene diol-epoxides with linear and supercoiled DNA.

Previous spectroscopic studies of the major adduct formed by reaction of (+/-)-7r,8t-dihydroxy-9t, 10t-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE-I) with linear DNA have been interpreted to suggest that the adduct is not intercalated in the double helix. However, studies of the electrophoretic mobility of supercoiled DNA treated with BPDE-I suggest that the adduct is intercalated. To resolve these interpretations, we have studied the reaction of BPDE-I with supercoiled and linear DNA. The kinetics of DNA-catalyzed hydrolysis and of covalent binding are similar for the two DNAs; supercoiled DNA exhibits a 20% increase in the rate of hydrolysis of BPDE-I at low DNA concentration compared to linear DNA. Fluorescence excitation spectra and fluorescence quenching experiments provide no support for a model in which BPDE-I adducts are intercalated in supercoiled DNA. When deoxyribonucleoside adducts were analyzed by high-performance liquid chromatography, identical distributions of BPDE-I adducts were found for supercoiled and linear DNA. These data are consistent with a previously proposed model (Hogan, M. E., Dattagupta, N., and Whitlock, J.P., Jr. J. Biol. Chem., 256: 4504-4513, 1981; Taylor, E.R., Miller, K. J., and Bleyer, A. J. J. Biomol. Struct. Dyn., 1: 883-904, 1983), in which the major BPDE-I adduct in both linear and supercoiled DNA exists in a conformation which allows stacking with the neighboring base pair and introduces a "kink" into the path of the helical axis. Although this model provides an explanation for all available experimental data, there are undoubtedly other DNA adduct conformational models which are also consistent with the data.

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

PubMed1986

Linearization of donor DNA during plasmid transformation in Neisseria gonorrhoeae.

We examined the fate of plasmid DNA after uptake during transformation in Neisseria gonorrhoeae. An 11.5-kilobase plasmid, pFA10, was processed to linear double-stranded DNA during uptake by competent cells, but cleavage of pFA10 was not site specific. A minority of pFA10 entered as open circles. A 42-kilobase plasmid, pFA14, was degraded into small fragments during uptake; no intracellular circular forms of pFA14 were evident. Since pFA10 DNA linearized by a restriction enzyme was not further cut during uptake, the endonucleolytic activity associated with entry of plasmid DNA appeared to act preferentially on circular DNA. Although linear plasmid DNA was taken up into a DNase-resistant state as efficiently as circular DNA, linear plasmid DNA transformed much less efficiently than circular plasmid DNA. These data suggest that during entry transforming plasmid DNA often is processed to double-stranded linear molecules; transformants may arise when some molecules are repaired to form circles. Occasional molecules which enter as intact circles may also lead to transformants.

DNA, Bacterial↗

PubMed1992

Mutational analysis by a combined application of the multiple restriction fragment-single strand conformation polymorphism and the direct linear amplification DNA sequencing protocols.

We describe here an improved procedure for polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) for rapid mutational detection. To circumvent the restriction of having to analyze relatively short PCR fragments, restriction endonucleases were used to cleave a longer PCR product and the mixture of fragments was analyzed directly in SSCP gel electrophoresis. This multiple restriction fragment (MRF)-SSCP protocol was demonstrated by the detection of a 4-bp deletion in codons 41-42 and a point mutation in the IVS-2 sequence of the human beta-globin gene. The MRF-SSCP or the standard SSCP protocol was then combined with the linear amplification DNA sequencing (LADS) procedure for direct analysis of the PCR products without further purification for an exact characterization of the mutations detected. In the LADS analysis, homo- or heterozygosity of a mutation was easily distinguished by the appearance of a single- or double-lane band in the sequencing gel. The choice of isotope used and different labeling methods were compared and were found, in some cases, to produce SSCP patterns of different complexities. The combined MRF-SSCP/LADS protocol permits rapid mutational analysis of a large number of clinical samples using only very small amounts of materials and can easily be adopted for nonisotopic clinical applications.

Base Sequence↗

PubMed1991

Interaction of the UvrABC endonuclease with DNA containing a psoralen monoadduct or cross-link. Differential effects of superhelical density and comparison of preincision complexes.

The effect of negative supercoiling on UvrABC incision of covalently closed duplex DNA circles containing either a furan-side monoadduct or a cross-link of 4'-hydroxymethyl-4,5',8-trimethylpsoralen at a unique site was examined. The rate of UvrABC incision of these DNA substrates was measured as a function of superhelical density, sigma, for values of sigma between 0 and -0.050. The monoadducted DNA substrate was incised at close to the maximum rate at all superhelical densities, with only a slight stimulation of activity between sigma = 0 and -0.035. In contrast, efficient UvrABC incision of the cross-linked DNA substrate required the DNA to be underwound, and activity showed a linear dependence on superhelical density up to sigma = -0.035. DNase I protection studies show that in the presence of both UvrA and UvrB a protein complex binds to the site of a psoralen monoadduct or cross-link in linear DNA. This UvrA-UvrB-dependent complex binds with similar affinity to both the monoadducted and the cross-linked DNA helices. However, differences in the DNase I footprint on these two DNA substrates indicate that the interaction of this protein complex is different at these two lesions. The addition of UvrC to linear DNA molecules that are saturated at the site of the lesion with the UvrA-UvrB-dependent complex resulted in efficient nicking of the monoadducted DNA, but not the cross-linked DNA. Thus, the properties of a DNA lesion site that lead to UvrAB recognition and binding are not necessarily sufficient to allow incision when all three Uvr subunits are present. We propose that after recognition and binding of a lesion site by the UvrAB complex and prior to incision, the damaged DNA helix undergoes a conformational change such as unwinding or melting that is induced by the lesion-bound Uvr complex.

Autoradiography↗

PubMed1989

Molecular dosimetry of hepatic aflatoxin B1-DNA adducts: linear correlation with hepatic cancer risk.

An analysis of hepatic aflatoxin B1-DNA adduct (HADA) concentrations in rats and trout demonstrated that the hepatic cancer risk was linearly and quantitatively related in both species. Additionally, the adduct-risk correlation for short-term exposure accurately predicted chronic exposure risk in trout. Although rat and trout liver cells differ substantially in their nuclear DNA content, they appeared to experience similar cancer risks at a given HADA concentration. This may indicate that the liver nuclei of both species contain a similar number of an equivalent protooncogene(s). This analysis also provided a possible basis for the use of adduct measurements for estimating human cancer risk. The minimum human virtual safe dose for aflatoxin was estimated to be 0.264 ng/kg/day on the basis of this approach.

Aflatoxin B1↗

PubMed1989

Presynapsis and synapsis of DNA promoted by the STP alpha and single-stranded DNA-binding proteins from Saccharomyces cerevisiae.

We previously purified an activity from meiotic cell extracts of Saccharomyces cerevisiae that promotes the transfer of a strand from a duplex linear DNA molecule to complementary circular single-stranded DNA, naming it Strand Transfer Protein alpha (STP alpha) (Sugino, A., Nitiss, J., and Resnick, M. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3683-3687). This activity requires no nucleotide cofactor but is stimulated more than 10-fold by the addition of yeast single-stranded DNA-binding proteins (ySSBs). In this paper, we describe the aggregation and strand transfer of double-stranded and single-stranded DNA promoted by STP alpha and ySSB. There is a good correlation between the aggregation induced by various DNA-binding proteins (ySSBs, DBPs and histone proteins) and the stimulation of STP alpha-mediated DNA strand transfer. This implies that the stimulation by ySSBs and other binding proteins is probably due to the condensation of single-stranded and double-stranded DNA substrates into coaggregates. Within these coaggregates there is a higher probability of pairing between homologous double-stranded and single-stranded DNA, favoring the initiation of strand transfer. The aggregation reaction is rapid and precedes any reactions related to DNA strand transfer. We propose that condensation into coaggregates is a presynaptic step in DNA strand transfer promoted by STP alpha and that pairing between homologous double- and single-stranded DNA (synapsis) occurs in these coaggregates. Synapsis promoted by STP alpha and ySSBs also occurs between covalently closed double-stranded DNA and single-stranded linear DNA as well as linear double-stranded and linear single-stranded DNAs in the absence of any nucleotide cofactors.

Catalysis↗

PubMed2005

Evaluation of the activity of HIV-1 integrase over-expressed in eukaryotic cells.

Since the integration of viral DNA in the host genome is an essential step in the replication cycle of HIV-1, an active search for inhibitors of the integration step is ongoing. Our laboratory has been working on the development of a cellular integration system. Such a system would be helpful in the study of the HIV-1 integration process and, eventually, could be used in the search for new inhibitors that selectively interfere with HIV integration. We have previously selected stable cell lines (293T-INS) that constitutively express high levels of HIV-1 integrase (IN) from a synthetic gene [FASEB J. 14 (2000) 1389]. We have now constructed linear DNA substrates containing the terminal HIV LTR sequences (so called 'mini-HIV') and EGFP as reporter gene to evaluate whether IN can improve the integration of transfected linear DNA. After electroporation of this mini-HIV we observed a 2- to 3-fold increase in EGFP expression in IN expressing cell lines relative to control cells. The increase in EGFP expression was still evident after passaging of the cells. The effect was observed with linear DNA but not with circular DNA, thus excluding an effect on DNA uptake. The increase was the highest in the 293T-INS(D64V) cell line due to an increase in the amount of total mini-HIV DNA and 2-LTR circles as quantified by Q-PCR. Our data suggest that IN over-expressed in our cell lines interacts with the incoming DNA, protects it from nuclease degradation but does not catalyze the integration as such.

Aspartic Acid↗